Respiratory Syncytial Virus (RSV) is a common cold virus belonging to the family of paramyxovirus. RSV is virulent, easily transmissible and the most common cause of lower respiratory tract disease in children of less than 2 years of age. Up to 98% of children attending day care will be infected in a single RSV season. Between 0.5% and 3.2% of children with RSV infection require hospitalization. Approximately 90,000 hospital admissions and 4500 deaths per year were reported in United States. Major risk factors for hospitalization due to RSV are premature birth, chronic lung disease, congenital heart disease, compromised immunity, and age younger than 6 weeks in otherwise healthy children.
Two subtypes of RSV have been identified, subtype A and subtype B. RSV has two major surface glycoproteins, the fusion protein (F protein) and the attachment protein (G protein). The F protein of RSV is a viral membrane protein and responsible for fusion of the virion with a host cell after attachment. In addition, infection of neighboring cells through the formation of syncytia is promoted by the F protein and its function is thought to depend on the original oligomeric structure of the protein. The G protein is a 89 kD protein which is also known as the attachment protein. The G protein differs considerably between the two RSV subtypes, whereas the F protein is more conserved. Approximately 53% homology is present within a G protein from subtypes A and B. Although G protein is not required for infection of host cells, anti-RSV G antibodies have shown to improve symptoms in animal models and can induce virus neutralization in the presence of complement.
Antibodies against the F or G protein of RSV have been described. Palivizumab is a genetically engineered, humanized monoclonal antibody against the F protein. WO 2008/147196 discloses sequences of human RSV F protein binding molecules. A mouse monoclonal antibody (131-2G) against the G protein has been described which is thought to bind to a CX3C (fractalkine) motif in the RSV G protein, which motif is capable of binding to the CX3CR1 (Fractalkine) receptor on NK cells, T cells and monocytes. This antibody was demonstrated to reduce migration of PBMC's towards RSV G glycoprotein (Tripp et al., 2001, Nat Immunol. 2001, 2(8):732-8). Antibody 131-2G does not neutralize RSV in vitro, however, in an in vivo mouse model dosing at 300 mg/mouse resulted in reduced RSV A2 recovery from lungs, reduced pulmonary inflammation, and lowered IFN-gamma levels in a mouse model. Human monoclonal antibodies against RSV G protein have been described in US 2010-0285022, WO 2009/055711 and Collarini et al. (Journal of Immunology, 2009, 183: 6338-6345). The antibodies bind to a conserved epitope in the G protein close to the CX3C domain, which is located in a region of the G protein corresponding to amino acid positions 164-172.
No effective treatment of RSV positive bronchiolitis beside supportive care in the form of adequate nutrition and oxygen therapy is currently available. Antiviral therapies such as Ribavirin have not been proven to be effective in RSV infection. Only monoclonal antibody palivizumab (also called Synagis), is registered for prophylaxis against RSV infection. However, palivizumab is not always effective. It is only useful and approved for prophylactic treatment of premature infants up to 4 KG body weight. Thus, palivizumab can not be used to treat an established RSV infection. Furthermore, palivizumab is only partly effective as it reduces hospitalization of infants by approximately 50%.
Therefore, there is a need for additional antibodies and therapies against RSV.
It is an object of the present invention to provide additional antibodies against the G protein of RSV, or functional equivalents of such antibodies and compositions comprising antibodies. Preferably antibodies are provided which recognize a different epitope as compared to known RSV antibodies. It is a further object to provide antibodies against the G protein of RSV, which are able to potentiate neutralizing activity of an antibody capable of binding an F protein of RSV.
The invention therefore provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof, capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein, wherein the numbering of amino acids is based on the RSV G protein of subtype A2 and B1 as depicted in FIG. 1.
A “functional part of an antibody” is defined as a part which has at least one shared property as said antibody in kind, not necessarily in amount. Said functional part is capable of binding the same antigen or epitope as said antibody, albeit not necessarily to the same extent. A functional part of an antibody preferably comprises a single domain antibody, a single chain antibody, a nanobody, an unibody, a single chain variable fragment (scFv), a Fab fragment or a F(ab′)2 fragment.
A functional part of an antibody is also produced by altering an antibody such that at least one property—preferably an antigen-binding property—of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is likely not to be seriously affected.
A “functional equivalent of an immunoglobulin chain” is defined herein as an artificial binding compound, comprising at least one CDR sequence of an immunoglobulin chain.
The term “a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof” includes isolated human antibodies or functional parts or immunoglobulins or functional equivalents thereof, as well as synthetic or recombinant antibodies or functional parts or immunoglobulins or functional equivalents thereof, the sequence of which is derived from the sequence of human antibodies.
Isolated, synthetic or recombinant antibodies or functional parts thereof, or immunoglobulin chains or functional equivalents thereof, capable of binding to a G protein of Respiratory Syncytial Virus described herein are also referred to as “RSV G-specific antibodies according to the invention”.
An RSV G-specific antibody according to the invention is preferably a human antibody. The use of human antibodies for prophylaxis and therapy in humans diminishes the chance of side-effects due to an immunological reaction in a human individual against non-human sequences. In another embodiment an RSV G-specific antibody according to the invention is a humanized antibody. Humanized antibodies are made by incorporating non-human hypervariable domains into human antibodies and therefore immunogenic properties are diminished as compared to fully non-human antibodies. In another embodiment an RSV G-specific antibody according to the invention is a chimeric antibody. In a chimeric antibody, sequences of interest, such as for instance a binding site of interest, are included into an RSV G-specific antibody according to the invention. FIG. 1 shows the amino acid sequence of RSV G protein of subtypes A2 and B1. If a part of RSV G protein is indicated herein by the amino acid residues of which said part consists, the numbering is based on the numbering shown in FIG. 1 and includes corresponding amino acid residues in G proteins of other RSV strains.
In one embodiment RSV G-specific antibodies according to the invention are capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. This provides the advantage that they bind to a different epitope as compared to previously disclosed RSV G antibodies. For instance, RSV G antibodies disclosed in US 2010-0285022 bind to multiple but different epitopes of the G protein of RSV which epitopes are located between amino acids 160-176 of the G protein. The CX3C motif is located between amino acids 182-186 as is shown in FIG. 1. Thus several preferred RSV G-specific antibodies according to the invention bind a different epitope of the G protein than the epitope of antibodies disclosed in US 2010-0285022 and the CX3C motif. RSV G-specific antibodies according to the invention that bind to a different epitope as compared to known RSV G antibodies are thus advantageously combined with such known antibodies in order to improve the treatment with antibodies. Such RSV G-specific antibodies according to the invention and such known antibodies do not compete for the same epitope in the G protein.
An RSV G-specific antibody according to the invention capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein is particularly suitable for combination with one or more known RSV G binding antibodies. Such preferred RSV G-specific antibodies according to the invention are also particularly suitable for combination with one or more other RSV G-specific antibodies according to the invention that are capable of binding other epitopes of RSV G protein, such as for instance a conformational epitope or an epitope which comprises the CX3C motif of the RSV G protein.
RSV G-specific antibodies (SEQ ID NOs: 294 and 295) according to the invention which are capable of binding an epitope of a G protein of RSV (SEQ ID NOs: 289-293) which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein, and thus are particularly preferred, are the antibodies designated AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 and AT50, which have heavy chain sequences of SEQ ID NO:133, 115, 116, 119, 125, 122, 110, 111, 114 and 124 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NO:2, 3, 5, 6, 7, 8, 11, 14, 16 and 17 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NO:20, 21, 23, 24, 25, 26, 29, 32, 34 and 35 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NO:38, 39, 41, 42, 43, 44, 47, 50, 52 and 53 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NO:56, 57, 59, 60, 61, 62, 65, 68, 70 and 71 being the light chain CDR1 sequences of these antibodies, SEQ ID NO:74, 75, 77, 78, 79, 80, 83, 86, 88 and 89 being the light chain CDR2 sequences of these antibodies, and SEQ ID NO:92, 93, 95, 96, 97, 98, 101, 104, 106 and 107 being the light chain CDR3 sequences of these antibodies.
The invention thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:
a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:2, 3, 5, 6, 7, 8, 11, 14, 16 and 17, and/or
a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 20, 21, 23, 24, 25, 26, 29, 32, 34 and 35, and/or
a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 38, 39, 41, 42, 43, 44, 47, 50, 52 and 53, and/or
a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 56, 57, 59, 60, 61, 62, 65, 68, 70 and 71, and/or
a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 74, 75, 77, 78, 79, 80, 83, 86, 88 and 89, and/or
a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 92, 93, 95, 96, 97, 98, 101, 104, 106 and 107. Preferably, said antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100% identical to these sequences.
Of course, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent according to the invention thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of the same antibody provided by the invention (as depicted in table 1).
The terms “AT35”, “AT37”, “AT39”, “AT43”, “AT51”, “AT47”, “AT32”, “AT33”, “AT36” and “AT50” as used herein encompass all antibodies and functional equivalents with the indicated heavy chain and light chain sequences, for instance isolated and/or purified or recombinantly produced. The indicated particularly preferred antibodies do not compete with antibody 3D3 described in US 2010-0285022 or monoclonal antibody 131-2G which binds to the CX3C motif in the G protein, which is located at amino acid positions 173-186 of the G protein. Thus, the indicated preferred antibodies are advantageously combined with these known antibodies.
In another embodiment, the invention provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part, or immunoglobulin chain or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV.
“An antibody capable of binding an F protein of RSV” is herein also called an RSV F-specific antibody. With the term “potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV” is meant that the RSV neutralizing activity of said antibody capable of binding an F protein of RSV is increased if an RSV G-specific antibody according to the invention is also present. An RSV G-specific antibody according to the invention is itself not capable of neutralizing RSV in the absence of complement factors. However, it was surprisingly found that the neutralizing activity of a RSV F-specific antibody is nevertheless increased if such RSV G-specific antibody according to the invention is present. Said neutralizing activity can be neutralizing activity in vitro or in vivo. An antibody capable of binding a F protein of RSV of which RSV neutralizing activity is potentiated by an RSV G-specific antibody according to the invention is preferably palivizumab, AM14, AM16, AM23 or D25, which are described in WO 2008/147196, or AM22, described in WO 2011/043643 and of which the heavy and light chain and CDR sequences are depicted in table 1.
Preferred RSV G-specific antibodies capable of potentiating RSV neutralizing activity of an RSV F-specific antibody are AT46, AT32, AT33 and AT35, which have heavy chain sequences of SEQ ID NO:109, 110, 111 and 113 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:127, 128, 129 and 131 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NO:1, 2, 3 and 5, being the heavy chain CDR1 sequences of these antibodies, SEQ ID NO:19, 20, 21 and 23 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NO:37, 38, 39 and 41 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NO:55, 56, 57 and 59 being the light chain CDR1 sequences of these antibodies, SEQ ID NO:73, 74, 75 and 77 being the light chain CDR2 sequences of these antibodies, and SEQ ID NO:91, 92, 93 and 95 being the light chain CDR3 sequences of these antibodies.
The invention thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:
a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:1, 2, 3 and 5, and/or
a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:19, 20, 21 and 23, and/or
a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:37, 38, 39 and 41, and/or
a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:55, 56, 57 and 59, and/or
a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:73, 74, 75 and 77, and/or
a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:91, 92, 93 and 95. Preferably, said antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100% identical to these sequences. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. Since AT46, AT32, AT33 and AT35 are preferred examples of antibodies capable of potentiating the RSV neutralizing activities of antibodies capable of binding an F protein of RSV, the invention thus provides a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part or immunoglobulin or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV, said antibody or functional part or immunoglobulin or functional equivalent having a combination of CDR sequences selected from the group consisting of:
SEQ ID NO: 1 (heavy chain CDR1 of AT46) and SEQ ID NO: 19 (heavy chain CDR2 of AT46) and SEQ ID NO: 37 (heavy chain CDR3 of AT46) and SEQ ID NO: 55 (light chain CDR1 of AT46) and SEQ ID NO: 73 (light chain CDR2 of AT46) and SEQ ID NO: 91 (light chain CDR3 of AT46); and
SEQ ID NO: 2 (heavy chain CDR1 of AT32) and SEQ ID NO: 20 (heavy chain CDR2 of AT32) and SEQ ID NO: 38 (heavy chain CDR3 of AT32) and SEQ ID NO: 56 (light chain CDR1 of AT32) and SEQ ID NO: 74 (light chain CDR2 of AT32) and SEQ ID NO: 92 (light chain CDR3 of AT32); and
SEQ ID NO:3 (heavy chain CDR1 of AT33) and SEQ ID NO: 21 (heavy chain CDR2 of AT33) and SEQ ID NO: 39 (heavy chain CDR3 of AT33) and SEQ ID NO: 57 (light chain CDR1 of AT33) and SEQ ID NO: 75 (light chain CDR2 of AT33) and SEQ ID NO: 93 (light chain CDR3 of AT33); and
SEQ ID NO: 5 (heavy chain CDR1 of AT35) and SEQ ID NO: 23 (heavy chain CDR2 of AT35) and SEQ ID NO: 41 (heavy chain CDR3 of AT35) and SEQ ID NO: 59 (light chain CDR1 of AT35) and SEQ ID NO: 77 (light chain CDR2 of AT35) and SEQ ID NO: 95 (light chain CDR3 of AT35); and
CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the sequences of any of these combinations.
In another preferred embodiment, the heavy and light sequences of one given antibody of interest (or sequences at least 70% identical thereto) are combined. Also provided is therefore a human isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin or functional equivalent thereof, capable of binding to a G protein of Respiratory Syncytial Virus (RSV), which antibody or functional part or immunoglobulin or functional equivalent is capable of potentiating RSV neutralizing activity of an antibody capable of binding an F protein of RSV, said antibody or functional part or immunoglobulin or functional equivalent having a combination of a heavy and light chain sequence selected from the group consisting of:
SEQ ID NO: 109 (heavy chain of AT46) and SEQ ID NO: 127 (light chain of AT46); and
SEQ ID NO: 110 (heavy chain of AT32) and SEQ ID NO: 128 (light chain of AT32); and
SEQ ID NO: 111 (heavy chain of AT33) and SEQ ID NO: 129 (light chain of AT33); and
SEQ ID NO: 113 (heavy chain of AT35) and SEQ ID NO: 131 (light chain of AT35); and
heavy and light chain sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the sequences of any of these combinations.
The terms “AT46”, “AT32”, “AT33” and “AT35” as used herein encompass all antibodies and functional equivalents with the indicated heavy chain and light chain sequences, for instance isolated and/or purified or recombinantly produced.
An advantage of a combination of an RSV F-specific antibody and an antibody according to the invention that is capable of potentiating RSV neutralizing activity of said RSV F-specific antibody is that a lower doses of said RSV F-specific antibody is needed in order to obtain the same neutralizing capacity. Therefore, less of said RSV F-specific antibody has to be administered to an individual for treatment and/or prevention of an RSV infection or RSV-related disorder. It is favourable to use an amount as low as possible to achieve a desired effect from both a health care of view, it is preferred to administer to a subject as less as possible of any substance, and from an economical point of view, a reduction of the amount of the therapeutic antibody needed generally reduces the cost of the treatment. Alternatively, with a similar amount of RSV F-specific antibody, a more effective treatment and/or prevention of an RSV infection and/or RSV-related disorder is achieved.
Furthermore, an RSV G-specific antibody according to the invention obviously recognizes a different epitope of RSV as an RSV F-specific antibody. By combining at least one RSV G-specific antibody according to the invention with an RSV F-specific antibody, two or more different targets in RSV are recognized during the same therapy. This way, a more potent anti-RSV treatment is obtained. Such a combination will result in more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
Furthermore, in a preferred embodiment, a lower overall antibody dosage is needed, as compared to current treatment with palivizumab. As already mentioned above, a lower amount of antibody capable of binding an F protein of RSV is needed to obtain the same neutralizing capacity. However, an RSV G-specific antibody according to the invention itself is also capable of counteracting RSV. Thus, in order to obtain a desired activity in counteracting RSV a lower total amount of (RSV G-specific and RSV F-specific) antibodies is needed if an RSV G-specific antibody according to the invention is combined with an RSV F-specific antibody.
An RSV G-specific antibody according to the invention capable of potentiating RSV neutralizing activity of an RSV F-specific antibody is thus advantageously combined with such an RSV F-specific antibody. Provided is thus a pharmaceutical composition comprising an RSV G-specific antibody according to the invention and an antibody capable of binding an F protein of RSV, and a pharmaceutically acceptable carrier, diluent and/or excipient. Such a pharmaceutical composition is particularly suitable for use in the treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
In the Examples, isolation of 17 antibodies according to the invention is described. The CDR's of these antibodies are depicted in table 1. The invention provides the insight that the CDR's with a sequence of SEQ ID NO:1-17, SEQ ID NO:19-35, SEQ ID NO:37-53, SEQ ID NO: 55-71, SEQ ID NO:73-89, and SEQ ID NO:91-107 provide particularly desired RSV binding characteristics. The invention therefore provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:
a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:1-17, and/or
a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:19-35, and/or
a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:37-53, and/or
a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:55-71, and/or
a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:73-89, and/or
a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:91-107.
Preferably, an RSV G-specific antibody according to the invention comprises a heavy and/or light chain CDR sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1-17, SEQ ID NO:19-35, SEQ ID NO:37-53, SEQ ID NO: 55-71, SEQ ID NO:73-89, and SEQ ID NO:91-107. Most preferably, an RSV G-specific antibody according to the invention comprises a heavy and/or light chain CDR sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to a sequence selected from the group consisting of SEQ ID NO:1-17, SEQ ID NO:19-35, SEQ ID NO:37-53, SEQ ID NO: 55-71, SEQ ID NO:73-89, and SEQ ID NO:91-107. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent according to the invention thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51.
Particularly preferred RSV G-specific antibodies according to the invention are the antibodies AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 and AT51, which have heavy chain and light chain CDR sequences as depicted in table 1, because these antibodies have been demonstrated to have particularly desired binding characteristics. In a preferred embodiment an RSV G-specific antibody according to the invention therefore comprises both the heavy and light chain CDR sequences of one of the above mentioned RSV G-specific antibodies.
Provided are thus RSV G-specific antibodies according to the invention which have heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT46, comprising the sequence of SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:37, SEQ ID NO:55, SEQ ID NO:73 and SEQ ID NO:91, or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT46 does not show competitive binding with any other antibody described herein and can thus be advantageously combined with any other RSV G-specific antibody described herein and known RSV G-specific antibodies. Antibody AT46 is also preferred because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus antibody AT46 has the advantage that is active against a wide range of RSV strains. Antibody AT46 is furthermore a particularly preferred antibody because it is capable of binding the G protein of both RSV A and B subtypes. Furthermore antibody AT46 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT46 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT32, comprising the sequence of SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:56, SEQ ID NO:74 and SEQ ID NO:92 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT32 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Antibody AT32 is also preferred because it is capable of binding an epitope of a G protein of RSV which epitope is RIPNK (amino acids 188-192) of said G protein, and has a high binding affinity, having an affinity constant (KD) of about 0.6 nM for the RSV Ga protein (Table 7a). Thus, antibody AT32 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT32 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. Furthermore antibody AT32 is capable of potentiating the RSV neutralizing activity of RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT32 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT33, comprising the sequence of SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:75 and SEQ ID NO:93 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT33 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.01 μg/ml. Antibody AT33 is also preferred because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT33 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT33 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein capable of binding the CX3C motif of the RSV G protein. Furthermore antibody AT33 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies, and can thus be advantageously combined with a RSV F-specific antibody, such as palivizumab, AM14, AM16, AM22, AM23 and D25. The characteristics of antibody AT33 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT34, comprising the sequence of SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76 and SEQ ID NO:94 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT34 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT34 is also preferred because it is capable of binding within or close to the conserved motif and/or the CX3C motif of the RSV G protein. Antibody AT34 can thus be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT34 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT35, comprising the sequence of SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:59, SEQ ID NO:77 and SEQ ID NO:95 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT35 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.08 μg/ml. Antibody AT35 is also preferred because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT35 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT35 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT35 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT36, comprising the sequence of SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:60, SEQ ID NO:78 and SEQ ID NO:96 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT36 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT36 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT36 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT36 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT37, comprising the sequence of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:79 and SEQ ID NO:97 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT37 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT37 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT37 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT37 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT39, comprising the sequence of SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:44, SEQ ID NO:62, SEQ ID NO:80 and SEQ ID NO:98 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT39 is a preferred antibody because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.10 μg/ml. Antibody AT39 is also preferred because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT39 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT39 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT39 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT40, comprising the sequence of SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:45, SEQ ID NO:63, SEQ ID NO:81 and SEQ ID NO:99 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT40 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Furthermore, AT40 has a high binding affinity, having an affinity constant (KD) of about 0.2 nM for RSV-Ga and about 0.1 nM for Gb as measured by IBIS-iSPR technology (Table 7a). Antibody AT40 is also preferred because it is capable of binding the epitope FEVFNF (SEQ ID NO: 297; amino acids 165-170) of the RSV G protein. Antibody AT40 can thus be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT40 are summarized in Tables 4, 5, 6 and 7.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT42, comprising the sequence of SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:46, SEQ ID NO:64, SEQ ID NO:82 and SEQ ID NO:100 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT42 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT42 is also preferred because it is capable of binding a conformational epitope of the RSV G protein, which domain is at least partially within the conserved domain (amino acids 164-172) and/or the CX3C binding domain (CWAIC; SEQ ID NO: 298) because AT42 competes with antibody 131-2G and partially competes with antibody 3D3 (Table 4 and FIG. 5). Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus antibody AT42 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT42 can be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Furthermore, AT42 has a high binding affinity, having an affinity constant (KD) of about 1.3 nM for RSV Ga and about 0.3 nM for Gb as measured by IBIS-iSPR technology (Table 7). The characteristics of antibody AT42 are summarized in Tables 4, 5, 6 and 7.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT43, comprising the sequence of SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:65, SEQ ID NO:83 and SEQ ID NO:101 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT43 is a preferred antibody because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus antibody AT43 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT43 can be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT43 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT44, comprising the sequence of SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:84 and SEQ ID NO:102 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT44 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.02 μg/ml. Furthermore, AT44 has a high binding affinity, having an affinity constant (KD) of about 0.1 nM for both RSV Ga and Gb as measured by IBIS-iSPR technology (Fable 7a and b). Antibody AT44 is also preferred because it is capable of binding the epitope EVFNF (SEQ ID NO: 299; amino acids 166-170) of the RSV G protein. Antibody AT44 can thus be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT44 are summarized in Tables 4, 5, 6 and 7.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT45, comprising the sequence of SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:49, SEQ ID NO:67, SEQ ID NO:85 and SEQ ID NO:103 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT45 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype and because it has a particularly high RSV neutralizing capacity, having an IC50 of about 0.11 μg/ml. Antibody AT45 is also preferred because it is capable of binding within or in the proximity of the CX3C motif of the RSV G protein. Antibody AT45 can thus be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT45 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT47, comprising the sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:50, SEQ ID NO:68, SEQ ID NO:86 and SEQ ID NO:104 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT47 is a preferred antibody because it is capable of binding a conformational epitope of the RSV G protein. Conformational epitopes are generally highly conserved within different RSV strains, as described in more detail herein elsewhere. Thus antibody AT47 has the advantage that is active against a wide range of RSV strains. Furthermore, because it binds a conformational epitope, antibody AT47 can be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to the CX3C motif of the RSV G protein and with RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT47 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT49, comprising the sequence of SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:51, SEQ ID NO:69, SEQ ID NO:87 and SEQ ID NO:105 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT49 is a preferred antibody because it is capable of binding the G protein of both RSV A and B subtype. Antibody AT49 is also preferred because it is capable of binding within or close to the CX3C motif of the RSV G protein. Antibody AT49 can thus be advantageously combined with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and RSV G-specific antibodies disclosed herein that are capable of binding an epitope of a G protein of Respiratory Syncytial Virus which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. The characteristics of antibody AT49 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT50, comprising the sequence of SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:70, SEQ ID NO:88 and SEQ ID NO:106 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT50 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT50 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT50 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and with RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT50 are summarized in Tables 4, 5 and 6.
In another embodiment an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences of antibody AT51, comprising the sequence of SEQ ID NO:17, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:71, SEQ ID NO:89 and SEQ ID NO:107 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto. Antibody AT51 is a preferred antibody because it is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Thus, antibody AT51 binds to a different epitope as compared to previously disclosed RSV G antibodies. Antibody AT51 can thus be advantageously combined with such known antibodies, with RSV G-specific antibodies disclosed herein that are capable of binding to a conformational epitope and RSV G-specific antibodies disclosed herein that are capable of binding the CX3C motif of the RSV G protein. The characteristics of antibody AT51 are summarized in Tables 4, 5 and 6.
Preferably, an RSV G-specific antibody according to the invention comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to the sequences of the same antibody of the invention as depicted in table 1.
As is well known by the skilled person, a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule. A heavy chain comprises constant domains and a variable domain, which variable domain is involved in antigen binding. A light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule. A light chain comprises a constant domain and a variable domain. The variable domain is, together with the variable domain of the heavy chain, involved in antigen binding.
Complementary-determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. The CDRs of a heavy chain and the connected light chain of an antibody together form the antigen-binding site.
Based on the human RSV G-specific antibodies depicted in table 1, it is possible to produce an immunoglobulin chain or functional equivalent thereof comprising at least one CDR sequence of a human immunoglobulin variable domain depicted in table 1 which is specific for RSV G protein. Further provided is thus an isolated, recombinant or synthetic immunoglobulin chain or functional equivalent thereof comprising at least one CDR sequence of a human immunoglobulin variable region depicted in table 1. In a preferred embodiment, a human antibody is provided because the use of a human antibody diminishes the chance of side-effects due to an immunological reaction in a human individual. Optionally, said at least one CDR sequence is optimized, preferably in order to improve binding efficacy or stability. This is for instance done by mutagenesis experiments where after the stability and/or binding efficacy of the resulting compounds are preferably tested and an improved RSV G-specific antibody is selected.
A skilled person is well capable of generating variants comprising at least one altered CDR sequence according to the invention. For instance, conservative amino acid substitution is applied. It is also possible to alter at least one CDR sequence depicted in table 1 in order to generate a variant antibody, or a functional part thereof, with at least one altered property as compared to the original antibody. Preferably, an antibody or functional part is provided comprising a CDR sequence which is at least 70% identical to a CDR sequence as depicted in table 1, so that the favorable binding characteristics of an RSV G-specific antibody according to the invention are at least in part maintained or even improved. A CDR sequence as depicted in table 1 is preferably altered such that the resulting antibody or functional part comprises at least one improved property, such as for instance an improved binding affinity, selectivity and/or stability, as compared to the original antibody. Variant antibodies or functional parts thereof comprising an amino acid sequence which is at least 70% identical to a CDR sequence as depicted in table 1 are therefore also within the scope of the present invention. Various methods are available in the art for altering an amino acid sequence. For instance, a heavy chain or light chain sequence with a desired CDR sequence is artificially synthesized. Preferably, a nucleic acid sequence encoding a CDR sequence according to the invention is mutated, for instance using random—or site-directed—mutagenesis.
Besides optimizing CDR sequences in order to improve binding efficacy or stability, it is often advantageous to optimize at least one sequence in at least one of the framework regions. This is preferably done in order to improve binding efficacy or stability. Framework sequences are for instance optimized by mutating a nucleic acid molecule encoding such framework sequence where after the characteristics of the resulting antibody—or functional part—are preferably tested. This way, it is possible to obtain improved antibodies or functional parts. In a preferred embodiment, human germline sequences are used for framework regions in antibodies or functional parts thereof or immunoglobulin chains or functional equivalents according to the invention. The use of germline sequences preferably minimizes the risk of immunogenicity of said antibodies or functional parts, immunoglobulin chains or functional equivalents, because these sequences are less likely to contain somatic alterations which are unique to individuals from which the framework regions are derived, and may cause an immunogenic response when applied to another human individual.
In a preferred embodiment, RSV G-specific antibodies according to the invention are provided that are capable of binding an epitope of a G protein of RSV which epitope is a non-linear or conformational epitope. The term “non-linear or conformational epitope” is herein defined as an epitope which is formed by the amino acid sequence and the three-dimensional shape of an antigen (e.g., folding). The amino acids making up the epitope can be relatively few in number and widely spread along the length of the molecule. Such epitope is brought into the correct conformation via folding of the antigen. In general, antibodies recognizing conformational epitopes afford broader specificity and therefore more effective therapeutic application for ameliorating or preventing RSV infection than antibodies able to bind only linear epitopes because conformational epitopes are more conserved. In order to obtain the necessary correct folding of a protein, variation within amino acids which are part of a conformational epitope is limited. Thus the antibodies capable of binding to a conformational epitope disclosed herein have the advantage that they are active against a wider range of RSV strains than antibodies recognizing linear epitopes. An RSV G-specific antibody according to the invention capable of binding a conformational epitope of a G protein of RSV is particularly suitable for combination with one or more RSV G-specific antibodies according to the invention capable of binding to another epitope, such as a linear epitope of RSV G protein which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of said G protein or an epitope which comprises the CX3C motif of the RSV G protein.
Particularly preferred RSV G-specific antibodies which bind to conformational epitopes are the antibodies designated AT46 AT42, AT43 and AT47 which have heavy chain sequences of SEQ ID NO:109, 118, 119 and 122 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:127, 136, 137 and 140 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NO:1, 10, 11 and 14 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NO:19, 28, 29 and 32 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NO:37, 46, 47 and 50 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NO:55, 64, 65 and 68 being the light chain CDR1 sequences of these antibodies, SEQ ID NO:73, 82, 83 and 86 being the light chain CDR2 sequences of these antibodies, and SEQ ID NO:91, 100, 101 and 104 being the light chain CDR3 sequences of these antibodies.
The invention thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:
a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 1, 10, 11 and 14, and/or
a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 19, 28, 29 and 32, and/or
a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 37, 46, 47 and 50, and/or
a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 55, 64, 65 and 68, and/or
a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 73, 82, 83 and 86, and/or
a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 91, 100, 101 and 104.
Preferably, said antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to these sequences. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent according to the invention thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT46 AT42, AT43 or AT47.
The terms “AT46”, “AT42”, “AT43” and “AT47” as used herein encompass all antibodies with the indicated heavy chain and light chain sequences, for instance isolated and/or purified or recombinantly produced.
The invention also provides RSV G-specific antibodies which are capable of binding to or close to the CX3C motif of the RSV G protein. An RSV G-specific antibody according to the invention capable of binding the CX3C motif of the RSV G protein is particularly suitable for combination with one or more RSV G-specific antibodies according to the invention capable of binding to another epitope, such as an epitope of RSV G protein which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of said G protein or an epitope capable of binding a conformational epitope of a G protein of RSV.
Particularly preferred RSV G-specific antibodies according to the invention which are capable of binding the CX3C motif of the RSV G protein are the antibodies designated AT34, AT40, AT49, AT44 and AT45 which have heavy chain sequences of SEQ ID NO:112, 117, 123, 120 and 121 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:130, 135, 141, 138 and 139 as depicted in table 1, respectively. The heavy and light chain CDR sequences of these preferred antibodies are also depicted in table 1, namely SEQ ID NO:4, 9, 12, 13 and 15 being the heavy chain CDR1 sequences of these antibodies, SEQ ID NO:22, 27, 30, 31 and 33 being the heavy chain CDR2 sequences of these antibodies, SEQ ID NO:40, 45, 48, 49 and 51 being the heavy chain CDR3 sequences of these antibodies, SEQ ID NO:58, 63, 66, 67 and 69 being the light chain CDR1 sequences of these antibodies, SEQ ID NO:76, 81, 84, 85 and 87 being the light chain CDR2 sequences of these antibodies, and SEQ ID NO:94, 99, 102, 103 and 105 being the light chain CDR3 sequences of these antibodies.
The invention thus provides an isolated, synthetic or recombinant antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof comprising:
a heavy chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 4, 9, 12, 13 and 15, and/or
a heavy chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 22, 27, 30, 31 and 33, and/or
a heavy chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 40, 45, 48, 49 and 51, and/or
a light chain CDR1 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 58, 63, 66, 67 and 69, and/or
a light chain CDR2 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 76, 81, 84, 85 and 87, and/or
a light chain CDR3 sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 94, 99, 102, 103 and 105. Preferably, said antibody or functional part or immunoglobulin chain or functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to these sequences. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. An antibody, functional part, immunoglobulin or functional equivalent according to the invention thus preferably comprises CDR sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3 sequences of antibody AT34, AT40, AT49, AT44 or AT45.
The terms “AT34”, “AT40”, “AT49”, “AT44” and “AT45” as used herein encompass all antibodies with the heavy chain and light chain sequences, for instance isolated and/or purified or recombinantly produced.
In a preferred embodiment, at least two RSV G-specific antibodies according to the invention are combined because with a combination of different antibodies RSV is more effectively counteracted. Particularly preferred is the combination of at least two RSV G-specific antibodies according to the invention which bind to different epitopes of the G protein. By combining at least two RSV G-specific antibodies which bind to different epitopes on the RSV G protein, two or more different epitopes of RSV G protein are recognized during the same therapy. This way, a more potent anti-RSV response is obtained. With a stronger response to RSV, such combination will result in more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder.
The invention therefore provides a composition comprising a combination of at least two RSV G-specific antibodies according to the invention. In a preferred embodiment, a composition according to the invention comprises at least two RSV G-specific antibodies selected from at least two of the following groups:
1) an RSV G-specific antibody according to the invention capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein. Preferred antibodies are AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 and AT50, which have heavy chain sequences of SEQ ID NO:113, 115, 116, 119, 125, 122, 110, 111, 114 and 124 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142 as depicted in table 1, respectively;
2) an RSV G-specific antibody according to the invention capable of binding a conformational epitope of an RSV G protein. Preferred antibodies are AT46, AT42, AT43 and AT47 which have heavy chain sequences of SEQ ID NO:109, 118, 119 and 122 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:127, 136, 137 and 140 as depicted in table 1, respectively; and
3) an RSV G-specific antibody according to the invention capable of binding the CX3C motif of the RSV G protein. Preferred antibodies are AT34, AT40, AT49, AT44 and AT45 which have heavy chain sequences of SEQ ID NO:112, 117, 123, 120 and 121 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:130, 135, 141, 138 and 139 as depicted in table 1, respectively.
A particularly preferred RSV G-specific antibody according to the invention is AT46, which has heavy and light chain sequences SEQ ID NO:109 and SEQ ID NO:127 as depicted in table 1. This antibody does not show competitive binding with any other antibody described herein. AT46 can thus be advantageously combined with any other RSV G-specific antibody described herein, including other antibodies which are capable of binding a conformational epitope, i.e. AT42, AT43 and AT47 which have heavy and light chain sequences as depicted in table 1. Thus, any combination of two RSV G-specific antibodies according to the invention which comprises at least AT46 is a combination of two antibodies binding to different epitopes of the RSV G protein. Antibody AT46 can thus be advantageously used in combination with any other RSV G-specific antibody according to the invention, and any known RSV G-specific antibody. Antibody AT46 is furthermore a particularly preferred antibody according to the invention because it is capable of binding the G protein of both RSV A and B subtypes and has a high binding affinity. Furthermore antibody AT46 is capable of potentiating the RSV neutralizing activity of several RSV F-specific antibodies. The characteristics of antibody AT46 are summarized in Tables 4, 5 and 6. Thus, in a preferred embodiment of the invention a composition comprises a combination of AT46 and an other RSV G-antibodies according to the invention.
Other preferred combinations of two RSV G-specific antibodies are depicted in tables 2 and 3. Therefore, in another preferred embodiment of the invention a composition comprises a combination of two RSV G-antibodies according to the invention wherein said combination is selected from table 2. More preferably, said combination is selected from table 3. One or more RSV G-specific antibodies according to the invention that are capable of binding a conformational epitope of RSV G protein are also advantageously combined with RSV G-specific antibodies that are already known, such as antibodies disclosed in US 2010-0285022. One or more RSV G-specific antibodies according to the invention are capable of binding to an epitope of RSV G protein which epitope is located between amino acids 51-158 and/or between amino acids 189-299 of said G protein are also advantageously combined with RSV G-specific antibodies that are already known, such as antibodies disclosed in US 2010-0285022.
The invention therefore also provides a composition comprising an RSV G-specific antibody according to the invention capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 51-160 and/or between amino acids 187-299 of said G protein (preferably antibody AT35, AT37, AT39, AT43, AT51, AT47, AT32, AT33, AT36 or AT50, which have heavy chain sequences of SEQ ID NO:113, 115, 116, 119, 125, 122, 110, 111, 114 and 124 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:131, 133, 134, 137, 143, 140, 128, 129, 132 and 142 as depicted in table 1, respectively), and a known RSV G-specific antibody which is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 164-186 of said G protein. Also provided is a composition comprising an RSV G-specific antibody according to the invention capable of binding a conformational epitope of an RSV G protein (preferably antibody AT46, AT42, AT43 or AT47 which have heavy chain sequences of SEQ ID NO:109, 118, 119 and 122 as depicted in table 1, respectively, and light chain sequences of SEQ ID NO:127, 136, 137 and 140 as depicted in table 1, respectively), and a known RSV G-specific antibody which is capable of binding an epitope of a G protein of RSV which epitope is located between amino acids 164-186 of said G protein.
Particularly preferred are combinations of three RSV G-specific antibodies according to the invention which bind to different epitopes of the G protein. By combining at least three of such RSV G-specific antibodies at least three different epitopes of RSV G protein are recognized during the same therapy. This way, often an even stronger immunogenic response to RSV is obtained and a higher antibody specificity against RSV is reached as compared to the use of one antibody or a combination of two antibodies. As indicated above, with a stronger immunogenic response to and/or a higher specificity against RSV, a more effective treatment and/or prevention of an RSV infection and/or an RSV-related disorder can be achieved. A combination of three RSV G-specific antibodies according to the invention preferably comprises three antibodies that do not compete for the same or overlapping epitopes in the RSV G protein. The invention therefore provides a composition comprising a combination of three RSV G-specific antibodies according to the invention, wherein said combination is selected from the group consisting of:                AT34+AT46+AT42;        AT40+AT46+AT42;        AT44+AT46+AT42;        AT45+AT46+AT42;        AT49+AT46+AT42;        
which antibodies have heavy and light chain sequences as depicted in table 1.
Other preferred combinations of three RSV G-specific antibodies according to the invention are AT34, AT33 and AT46; and AT36, AT46 and AT45. These combinations of three RSV G-specific antibodies according to the invention have been proven to be able to non-competitively bind the RSV G protein using IBIS-iSPR technology (IBIS Technologies BV Hengelo, the Netherlands).
Other preferred combinations of two or three antibodies are:                AT42+AT33;        AT42+AT44;        AT40+AT46+AT32;        AT40+AT46+AT33;        AT44+AT42+AT33;        AT44+AT46+AT33;        
These combinations of anti-RSV G antibodies were able to neutralize the virus without the addition of complement factors as demonstrated in example 2 and FIG. 3A.
Preferred RSV G-specific antibodies according to the invention are capable of binding the G protein of both RSV subtype A and RSV subtype B because such antibodies can be used for counteracting both RSV subtypes. However, RSV G-specific antibodies according to the invention capable of binding the G protein of RSV subtype A only are also particularly useful. For instance, RSV G-specific antibodies according to the invention that only bind the G protein of RSV subtype A, bind to a different epitope in the G protein of RSV than RSV G-specific antibodies described herein that bind to both subtype A and B RSV. Therefore, they are particularly suitable to be used in combination with RSV G-specific antibodies that bind to both subtype A and B RSV as described above. Furthermore, RSV G-specific antibodies according to the invention that only bind the G protein of RSV subtype A are particularly suitable for diagnosing RSV subtype A.
Preferred RSV G-specific antibodies according to the invention have a high affinity for the RSV G protein. Measurement of the affinity constant and specificity of binding between antigen and antibody is preferred in determining the efficacy of prophylactic, therapeutic, diagnostic and research methods using anti-RSV G antibodies of the invention. “Binding affinity” generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity can generally be represented by the equilibrium dissociation constant (KD), which is calculated as the ka to kd ratio (See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881, and Table 7 and FIG. 7). Affinity can be measured by common methods known in the art, such as for instance a surface plasmon resonance (SPR) assay such as BiaCore or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo, the Netherlands) or solution phase assays, such as Kinexa. Preferably an RSV G-specific antibody according to the invention has an affinity constant (KD) as measured by IBIS-iSPR technology of at most 10 nM, more preferably at most 5 nM, more preferably at most 2 nM, more preferably at most 1 nM, more preferably at most 0.5 nM, more preferably at most 0.3 nM, more preferably at most 0.1 nM.
Other preferred RSV G-specific antibodies according to the invention have a high RSV neutralizing activity in the presence of complement. RSV neutralizing activity is for instance determined in vitro in the presence of complement, for instance rabbit serum complement. Rabbit serum complement is a mixture of complement factors prepared from the serum of rabbits and is commercially available from for instance GTi Diagnostics or Calbiochem. An in vitro neutralization assay in the presence of complement is for instance performed as described in the Examples. Preferably an RSV G-specific antibody according to the invention is capable of neutralizing RSV in vitro in the presence of complement with an IC50<500 ng/ml, more preferably with an IC50<400 ng/ml, more preferably with an IC50<350 ng/ml, more preferably with an IC50>300 ng/ml, more preferably with an IC50<250 ng/ml, more preferably with an IC50<200 ng/ml, more preferably with an IC50<150 ng/ml, most preferably with IC50<125 ng/ml. Further provided is therefore an RSV G-specific antibody according to the invention which has an RSV neutralizing capacity in vitro in the presence of complement with an IC50<500 ng/ml, more preferably with an IC50<400 ng/ml, more preferably with an IC50<350 ng/ml, more preferably with an IC50>300 ng/ml, more preferably with an IC50<250 ng/ml, more preferably with an IC50<200 ng/ml, more preferably with an IC50<150 ng/ml, most preferably with IC50<125 ng/ml. In one embodiment, an RSV G-specific antibody according to the invention has RSV neutralizing capacity in vitro in the presence of complement with an IC50<100 ng/ml, such as <80 ng/ml, or <25 ng/ml.
In a preferred embodiment, an RSV G-specific antibody according to the invention comprises a heavy chain sequence and/or light chain sequence, or a sequence which has at least 70% sequence identity thereto, as depicted in table 1. Also provided is therefore an antibody or functional part, or immunoglobulin chain or functional equivalent, having a heavy chain sequence comprising a sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or having a light chain sequence which is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO:127-143.
Preferably, an RSV G-specific antibody according to the invention comprises a heavy chain sequence which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or a light chain which is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:127-143. Most preferably, an RSV G-specific antibody according to the invention comprises a heavy chain sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to a sequence selected from the group consisting of SEQ ID NO:109-125 and/or a light chain sequence which is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% identical to a sequence selected from the group consisting of SEQ ID NO:127-143. The higher the identity, the more closely an antibody resembles an antibody depicted in table 1.
An antibody or functional part or immunoglobulin chain or functional equivalent according to the invention preferably comprises a heavy chain as well as a light chain which resemble the heavy and the light chain of the same antibody depicted in table 1. Thus, in a preferred embodiment an RSV G-specific antibody according to the invention comprises a heavy chain sequence of a given antibody, preferably antibody AT46, comprising the sequence of SEQ ID NO:109 and a light chain sequence of the same antibody, preferably AT46, comprising the sequence of SEQ ID NO:127, or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical thereto.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT32, comprising the sequence of SEQ ID NO:110 and the light chain sequence of antibody AT32, comprising the sequence of SEQ ID NO:128.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT33, comprising the sequence of SEQ ID NO:111 and a light chain sequence comprising the sequence of SEQ ID NO:129.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT34, comprising the sequence of SEQ ID NO:112 and a light chain sequence of antibody AT34, comprising the sequence of SEQ ID NO:130.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT35, comprising the sequence of SEQ ID NO:113 and a light chain sequence of antibody AT35, comprising the sequence of SEQ ID NO:131.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT36, comprising the sequence of SEQ ID NO:114 and a light chain sequence of antibody AT36, comprising the sequence of SEQ ID NO:132.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT37, comprising the sequence of SEQ ID NO:115 and a light chain sequence of antibody AT37, comprising the sequence of SEQ ID NO:133.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT39, comprising the sequence of SEQ ID NO:116 and a light chain sequence of antibody AT39, comprising the sequence of SEQ ID NO:134.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT40, comprising the sequence of SEQ ID NO:117 and a light chain sequence of antibody AT40, comprising the sequence of SEQ ID NO:135.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT42, comprising the sequence of SEQ ID NO:118 and a light chain sequence of antibody AT42, comprising the sequence of SEQ ID NO:136.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT43, comprising the sequence of SEQ ID NO:119 and a light chain sequence of antibody AT43, comprising the sequence of SEQ ID NO:137.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT44, comprising the sequence of SEQ ID NO:120 and a light chain sequence of antibody AT44, comprising the sequence of SEQ ID NO:138.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT45, comprising the sequence of SEQ ID NO:121 and a light chain sequence of antibody AT45, comprising the sequence of SEQ ID NO:139.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT47, comprising the sequence of SEQ ID NO:122 and a light chain sequence of antibody AT47, comprising the sequence of SEQ ID NO:140.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT49, comprising the sequence of SEQ ID NO:123 and a light chain sequence of antibody AT49, comprising the sequence of SEQ ID NO:141.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT50, comprising the sequence of SEQ ID NO:124 and a light chain sequence of antibody AT50, comprising the sequence of SEQ ID NO:142.
In another embodiment an RSV G-specific antibody according to the invention or functional part thereof comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain sequence of antibody AT51, comprising the sequence of SEQ ID NO:125 and a light chain sequence of antibody AT51, comprising the sequence of SEQ ID NO:143.
Preferably, an RSV G-specific antibody according to the invention or functional part thereof comprises sequences that are at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to the heavy and light chain sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51 as depicted in table 1.
The invention further provides an isolated, synthetic or recombinant nucleic acid sequence with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody or functional part, or immunoglobulin chain or functional equivalent according to the invention. Preferably a nucleic acid according to the invention has a length of at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 75 nucleotides. A nucleic acid according to the invention is for instance isolated from a B-cell which is capable of producing an RSV G-specific antibody according to the invention. In a preferred embodiment a nucleic acid encoding an RSV G-specific antibody according to the invention is provided.
As used herein “an isolated, synthetic or recombinant nucleic acid sequence with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one CDR sequence of an antibody or functional part thereof, or immunoglobulin chain or functional equivalent thereof according to the invention” is herein also referred to as “a nucleic acid sequence or functional equivalent thereof according to the invention”.
As used herein, a nucleic acid molecule or nucleic acid sequence of the invention preferably comprises a chain of nucleotides, more preferably DNA and/or RNA. In other embodiments a nucleic acid molecule or nucleic acid sequence of the invention comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid structures are referred to as functional equivalents of a nucleic acid sequence. The term “functional equivalent of a nucleic acid sequence” also encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides.
Nucleic acid sequences encoding preferred heavy chain and light chain CDR's of antibodies AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 and AT51 are depicted in table 1. Nucleic acid sequences encoding a heavy or light chain CDR of a RSV G-specific antibody according to the invention which differ from the CDR nucleic acid sequences depicted in table 1 but have nucleic acid codons encoding for the same amino acids of said heavy or light chain CDR are also encompassed by the invention. Nucleic acid sequences encoding a heavy or light chain CDR of a RSV G-specific antibody depicted in table 1 which has been altered, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), are also encompassed by the invention, as long as the resulting CDR has at least 70% sequence identity with a CDR depicted in table 1.
A preferred nucleic acid sequence according to the invention comprises:                a heavy chain CDR1 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:145-161, and/or        a heavy chain CDR2 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:163-179, and/or        a heavy chain CDR3 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:181-197, and/or        a light chain CDR1 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:199-215, and/or        a light chain CDR2 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:217-233, and/or        a light chain CDR3 encoding sequence which has at least 70% sequence identity to a sequence which is selected from the group consisting of SEQ ID NO:235-251.        
A nucleic acid sequence according to the invention preferably comprises a sequence which has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity to said sequence. Preferably, said nucleic acid sequence comprises at least one CDR encoding sequence. Further provided is a nucleic acid sequence or functional equivalent thereof comprising a sequence which has at least 70% sequence identity, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:145-161, SEQ ID NO:163-179, SEQ ID NO: 199-215, SEQ ID NO: 199-215, SEQ ID NO: 217-233, and SEQ ID NO: 235-251, said nucleic acid sequence or functional equivalent having at least 15 nucleotides. As described before, the six CDR sequences of one given antibody of interest (or sequences at least 70% identical thereto) are typically combined. A preferred nucleic acid sequence according to the invention therefore comprises CDR encoding sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%, identical to the heavy chain CDR1, CDR2 and CDR3 encoding sequences and the light chain CDR1, CDR2 and CDR3 encoding sequences of antibody AT46, AT32, AT33, AT34, AT35, AT36, AT37, AT39, AT40, AT42, AT43, AT44, AT45, AT47, AT49, AT50 or AT51.
A nucleic acid sequence or functional equivalent thereof according to the present invention preferably encodes a region which has at least 70% sequence identity to a heavy chain and/or a light chain as depicted in table 1. Thus, a preferred nucleic acid sequence or a functional equivalent comprises a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:253-269 and/or a sequence which has at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NO:271-287. More preferably, a nucleic acid sequence or a functional equivalent according to the invention comprises a heavy chain encoding sequence as well as a light chain encoding sequence which resemble the heavy and the light chain encoding sequences of the same antibody depicted in table 1. Thus, in a preferred embodiment a nucleic acid or functional equivalent according to the invention comprises a heavy chain encoding sequence of antibody AT46, comprising the sequence of SEQ ID NO:253 and a light chain encoding sequence of antibody AT46, comprising the sequence of SEQ ID NO:271 or sequences that are at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical thereto.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT32, comprising the sequence of SEQ ID NO:254 and the light chain encoding sequence of antibody AT32, comprising the sequence of SEQ ID NO:272.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT33, comprising the sequence of SEQ ID NO:255 and a light chain encoding sequence comprising the sequence of SEQ ID NO:273.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT34, comprising the sequence of SEQ ID NO:256 and a light chain encoding sequence of antibody AT34, comprising the sequence of SEQ ID NO:274.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT35, comprising the sequence of SEQ ID NO:257 and a light chain encoding sequence of antibody AT35, comprising the sequence of SEQ ID NO:275.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT36, comprising the sequence of SEQ ID NO:258 and a light chain encoding sequence of antibody AT36, comprising the sequence of SEQ ID NO:276.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT37, comprising the sequence of SEQ ID NO:259 and a light chain encoding sequence of antibody AT37, comprising the sequence of SEQ ID NO:277.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT39, comprising the sequence of SEQ ID NO:260 and a light chain encoding sequence of antibody AT39, comprising the sequence of SEQ ID NO:278.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT40, comprising the sequence of SEQ ID NO:261 and a light chain encoding sequence of antibody AT40, comprising the sequence of SEQ ID NO:279.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT42, comprising the sequence of SEQ ID NO:262 and a light chain encoding sequence of antibody AT42, comprising the sequence of SEQ ID NO:280.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT43, comprising the sequence of SEQ ID NO:263 and a light chain encoding sequence of antibody AT43, comprising the sequence of SEQ ID NO:281.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT44, comprising the sequence of SEQ ID NO:264 and a light chain encoding sequence of antibody AT44, comprising the sequence of SEQ ID NO:282.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT45, comprising the sequence of SEQ ID NO:265 and a light chain encoding sequence of antibody AT45, comprising the sequence of SEQ ID NO:283.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT47, comprising the sequence of SEQ ID NO:266 and a light chain encoding sequence of antibody AT47, comprising the sequence of SEQ ID NO:284.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT49, comprising the sequence of SEQ ID NO:267 and a light chain encoding sequence of antibody AT49, comprising the sequence of SEQ ID NO:285.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT50, comprising the sequence of SEQ ID NO:268 and a light chain encoding sequence of antibody AT50, comprising the sequence of SEQ ID NO:286.
In another embodiment a nucleic acid or functional equivalent according to the invention comprises a sequence which has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably at least 100% sequence identity with the heavy chain encoding sequence of antibody AT51, comprising the sequence of SEQ ID NO:269 and a light chain encoding sequence of antibody AT51, comprising the sequence of SEQ ID NO:287.
The term “% sequence identity” is defined herein as the percentage of residues in a candidate amino acid of nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art.
Further provided is a vector comprising a nucleic acid sequence or functional equivalent according to the invention. As used herein “a vector comprising a nucleic acid sequence or functional equivalent according to the invention” is also referred to as “a vector according to the invention”. Methods for constructing a vector with a nucleic acid sequence or functional equivalent according to the invention are well known in the art. Non-limiting examples of vectors suitable for generating a vector of the invention are retroviral and lentiviral vectors. Such vector is suitable for a variety of applications. For instance, a vector of the invention comprising a therapeutically beneficial nucleic acid sequence is suitable for prophylactic or therapeutic applications. Administration of such vector to an individual, preferably a human, in need thereof results in expression of said prophylactic or therapeutic nucleic acid sequence in vivo. Said vector can also be used in applications involving in vitro expression of a nucleic acid sequence of interest, for instance for (commercial) production of antibodies or functional equivalents according to the invention. Also provided is therefore an isolated or recombinant cell comprising a nucleic acid sequence or functional equivalent a vector according to the invention.
A nucleic acid sequence or vector according to the present invention is particularly useful for generating antibodies or functional parts, or immunoglobulin chains or functional equivalents which are specific for RSV G protein. This is for instance done by introducing such nucleic acid sequence or vector into a cell so that the cell's nucleic acid translation machinery will produce the encoded antibodies or functional parts, or immunoglobulin chains or functional equivalents. In one embodiment, a nucleic acid sequence or vector encoding a heavy and/or light chain according to the invention is expressed in so called producer cells, such as for instance cells of a Chinese hamster ovary (CHO), NSO (a mouse myeloma) or 293(1) cell line, some of which are adapted to commercial antibody production. Proliferation of said producer cells results in a producer cell line capable of producing RSV G-specific antibodies according to the invention. Preferably, said producer cell line is suitable for producing antibodies for use in humans. Hence, said producer cell line is preferably free of pathogenic agents such as pathogenic micro-organisms. Most preferably, RSV G-specific antibodies consisting of human sequences are generated using at least one nucleic acid sequence or vector according to the invention.
An isolated or recombinant antibody producing cell capable of producing an RSV G-specific antibody according to the invention is therefore also provided. An antibody producing cell is defined herein as a cell which is capable of producing and/or secreting antibodies or functional equivalents thereof, and/or which is capable of developing into a cell which is capable of producing and/or secreting antibodies or functional equivalents thereof. An antibody producing cell according to the invention is preferably a producer cell which is adapted to commercial antibody production. Preferably, said producer cell is suitable for producing antibodies for use in humans. A method for producing an RSV G-specific antibody according to the invention is also provided, said method comprising providing a cell, preferably an antibody producing cell, with a nucleic acid sequence or functional equivalent, or a vector according to the invention, and allowing said cell to translate said nucleic acid sequence or functional equivalent, or vector, thereby producing RSV G-specific antibodies according to the invention. A method according to the invention preferably further comprises a step of harvesting, purifying and/or isolating RSV G-specific antibodies according to the invention. Obtained RSV G-specific antibodies according to the invention are preferably used in human therapy, optionally after additional purifying, isolation or processing steps.
In one embodiment, an RSV G-specific antibody according to the invention is coupled to another moiety to form an antibody-drug conjugate. An RSV G-specific antibody according to the invention is for instance coupled to an antiviral agent, such as acyclovir, penciclovar, lamivudine, ribavirin, zanamivir, laninamivir, peramivir, idoxuridine, amantadine, remantidine, maxamine or thymalfasin. The term “antiviral agent” as used herein refers to any substance that reduces or blocks the function, or growth, of a virus and/or causes destruction of a virus. In another embodiment, a moiety that is coupled to an RSV G-specific antibody according to the invention is an antimicrobial peptide. The term “antimicrobial peptide” as used herein refers to small amphipathic peptides of variable length (typically 6 to 100 aminoacids), sequence and structure with activity against microorganisms such as for instance bacteria, protozoa, yeast, fungi and/or virusses. Antimicrobial peptides usually act through relatively non-specific mechanisms resulting in membranolytic activity but several antimicrobial peptides can also stimulate the innate immune response. In a preferred embodiment, said antimicrobial peptide has anti-viral activity. Non-limiting examples of suitable antimicrobial peptides are magainins, PGLa, cathelicidins (such as LL-37 and cathelicidin-related antimicrobial peptide (CRAMP)), alamethicin, mellitin and cecropin, hydramacin-1, pexiganan, MSI-78, MSI-843, MSI-594, polyphemusin, human antimicrobial peptide, defensins, protegrins and indolicidin. In yet another embodiment, a moiety that is coupled to an RSV G-specific antibody according to the invention is an immunomodulatory molecule such as an CD3 antibody. Such CD3 antibody is capable of binding T cells and, if coupled to an RSV G-specific antibody according to the invention, targeting T cells to RSV infected cells.
Said other moiety, for example a cytotoxic agent, is preferably coupled to an RSV G-specific antibody according to the invention via a linker such as an acid-labile hydrazone linker, via a peptide linker like citruline-valine, through a thioether linkage, or by sortase catalized transamidation, which is described in detail in WO 2010/087994.
Sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG; SEQ ID NO: 300) on the heavy chain of an antibody, preferably on the C-terminal part of the heavy chain, and on the moiety to be coupled to said antibody. The antibody and the moiety further typically contain a GGGGS (SEQ ID NO: 301) sequence and a tag for purification purposes, such as a HIS tag. Subsequently sortase mediated transamidation is performed followed by click chemistry linkage. In a sortase catalized transaminidation, “click chemistry linkage” typically involves chemical coupling of, for instance, an alkyne-containing reagent and, for instance, an azide-containing reagent which are added by sortase through addition of glycines to the sortase motif on the heavy chain of the antibody and to a sortase motif on the moiety (such as a protein, peptide or antibody) to be coupled to the antibody. In one embodiment, the invention therefore provides an RSV G-specific antibody according to the invention wherein a sortase recognition site (LPETGG; SEQ ID NO: 300) is engineered on the heavy chain of the antibody, preferably on the C-terminal part of the heavy chain, the antibody further containing a GGGGS (SEQ ID NO: 301) sequence and a purification tag, such as a HIS tag.
In another embodiment an RSV G-specific antibody according to the invention is coupled to another moiety via a thioether linkage. In such case, one or more cysteines are preferably incorporated into an RSV G-specific antibody according to the invention. Cysteines contain a thiol group and, therefore, incorporation of one or more cysteines into, or replacement of one or more amino acids by one or more cysteines of an RSV G-specific antibody according to the invention enable coupling of said RSV G-specific antibody to another moiety. Said one or more cysteines are preferably introduced into an RSV G-specific antibody according to the invention at a position where it does not influence folding of said antibody, and does not alter antigen binding or effector function. The invention therefore also provides an RSV G-specific antibody according to the invention wherein at least one amino acid other than cysteine has been replaced by a cysteine.
As described herein before, an RSV G-specific antibody according to the invention, preferably AT46, AT32, AT33 or AT35, and an RSV F-specific antibody, such as palivizumab, AM14, AM16, AM23, AM22 or D25 can be advantageously used in combination. Furthermore, it is also advantageous to combine an RSV G-specific antibody according to the invention with another RSV G-specific antibody according to the invention recognizing a different epitope or with a known RSV G-specific antibody recognizing a different epitope. In another embodiment, however, the invention provides an RSV bispecific antibody with specificity for both an RSV G protein and an RSV F protein, or with specificity to different epitopes within an RSV G protein. An “RSV bispecific antibody” as used herein is defined as an antibody capable of simultaneously binding two different epitopes, which epitopes may be located within the same antigen, i.e. the RSV G protein, or located within different antigens, i.e. the RSV G and F protein, and is also referred to as “an RSV bispecific antibody according to the invention”. The term “RSV bispecific antibody” also encompasses functional parts of such RSV bispecific antibodies which has retained its capability of binding a least two different epitopes simultaneously, such as bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment. Also provided is a pharmaceutical composition comprising an RSV bispecific antibody according to the invention.
In one embodiment, a bispecific antibody according to the invention comprises two non-identical heavy chain-light chain combinations, thus having two antigen-binding regions which recognize two different epitopes within the RSV G protein or which recognize one epitope in an RSV G protein and one epitope within an RSV F protein. For instance, in one embodiment, an RSV bispecific antibody comprises a heavy and light chain of an RSV G-specific antibody according to the invention as depicted in table 1 and a heavy and light chain of another RSV G-specific antibody according to the invention as depicted in table 1. In another embodiment, an RSV bispecific antibody comprises a heavy and light chain of an RSV G-specific antibody according to the invention as depicted in table 1 and a heavy and light chain of an RSV F-specific antibody. Bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment comprise for instance a scFv or Fab fragment of an RSV G-specific antibody according to the invention and a scFv or Fab fragment of another RSV G-specific antibody according to the invention. Alternatively, bispecific single chain variable fragments (scFv), bispecific Fab fragments and a bispecific F(ab′)2 fragment comprise a scFv or Fab fragment of an RSV G-specific antibody and a scFv or Fab fragment of an RSV F-specific antibody. In a preferred embodiment, an RSV bispecific antibody according to the invention comprises a heavy and light chain of antibody AT46, AT32, AT33 or AT35 as depicted in Table 1 or a scFv or Fab fragment thereof, and a heavy and light chain of an RSV F-specific antibody such as palivizumab, AM14, AM16, AM23, D25 (WO 2008/147196), or AM22 (WO 2011/043643) or a scFv or Fab fragment thereof. In another preferred embodiment, an RSV bispecific antibody according to the invention comprises two heavy and light chains of two different RSV G-specific antibodies according to the invention as depicted in Table 1, or a scFv or Fab fragment thereof, whereby said different RSV G-specific antibodies preferably form a combination depicted in Table 2 or 3.
In another embodiment, an RSV G-specific antibody according to the invention is coupled to an RSV F-specific antibody or another RSV G-specific antibody by sortase catalized transamidation, which is described herein before and in detail in WO 2010/087994. For this purpose, sortase catalized transamidation involves engineering of a sortase recognition site (LPETGG; SEQ ID NO: 300) on heavy chain of both antibodies to be coupled, preferably on the C-terminal part of the heavy chain. The antibodies further typically contain a GGGGS (SEQ ID NO: 301) sequence and a purification tag, such as a HIS tag. Thus, if an RSV G-specific antibody according to the invention and an RSV F-specific antibody are coupled, both said RSV G-specific and said RSV F-specific antibodies are engineered as described herein before and in detail in WO 2010/087994. If two RSV G-specific antibodies recognizing different epitopes in the G protein are coupled, both said RSV G-specific antibodies are engineered as described herein before and in detail in WO 2010/087994. Subsequently sortase mediated transamidation is preferably performed followed by click chemistry linkage to couple both antibodies via their heavy chains. As herein explained before, “click chemistry linkage” involves chemical coupling of, for instance, an alkyne-containing reagent and, for instance, an azide-containing reagent which are added by sortase through addition of glycines to the sortase motif on the heavy chain of a first antibody and to the heavy chain of a second antibody to be coupled to the first antibody. In a preferred embodiment, antibody AT46, AT32, AT33 or AT35 as depicted in Table 1 is coupled by sortase catalized transamidation to an RSV F-specific antibody, such as palivizumab, AM14, AM16, AM23, D25, or AM22. In another preferred embodiment, two RSV G-specific antibodies are coupled to each other by sortase catalized transamidation, whereby said RSV G-specific antibodies preferably form a combination depicted in Table 2 or 3.
RSV G-specific antibodies according to the invention are capable of counteracting Respiratory Syncytial Virus. RSV G-specific antibodies according to the invention are therefore particularly suitable for use as a medicine or prophylactic agent. Preferably, RSV G-specific antibodies according to the invention are used which consist of human sequences, in order to reduce the chance of adverse side effects when human individuals are treated. Such human sequences can be isolated from a human or synthetically or recombinantly produced based on the sequence of human antibodies Provided is therefore an RSV G-specific antibody according to the invention or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention for use as a medicament and/or prophylactic agent. Also provided is a nucleic acid sequence or functional equivalent thereof according to the invention or a vector according to the invention comprising such nucleic acid or functional equivalent for use as a medicament and/or prophylactic agent. When a nucleic acid or functional equivalent according to the invention is administered, it will be translated in situ by the host's machinery into an RSV G-specific antibody according to the invention. Produced RSV G-specific antibodies according to the invention are capable of preventing and/or counteracting an RSV infection or RSV related disorder. RSV G-specific antibodies according to the invention are particularly suitable for use as a medicament because they are capable of counteracting RSV after an individual has been infected. On the contrary, palivizumab, the only anti-RSV antibody currently registered, is only useful for prophylactic treatment of premature infants and is thus not able to treat an established RSV infection. In a particularly preferred embodiment said antibody comprises antibody AT46, or a functional part thereof. Provided is thus antibody AT46, comprising a heavy chain sequence of SEQ ID NO:109 and a light chain sequence of SEQ ID NO:127, for use as a medicament and/or prophylactic agent.
An RSV G-specific antibody according to the invention, or a nucleic acid sequence or functional equivalent thereof according to the invention, or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention, or an RSV bispecific antibody or a cell according to the invention is preferably used for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder. As used herein “at least in part treating an RSV infection” includes counteracting an RSV infection, alleviating symptoms resulting from an RSV infection and/or counteracting inflammation resulting from an RSV infection. Also provided is therefore an RSV G-specific antibody according to the invention, or a nucleic acid sequence or functional equivalent thereof according to the invention, or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention, or a vector according to the invention, or a cell according to the invention, or a RSV bispecific antibody according to the invention, for use in a method of at least in part treating and/or preventing an RSV infection and/or an RSV related disorder. Examples of such RSV related disorders are bronchiolitis, pneumonia and tracheobronchitis resulting from an RSV infection. Further provided is a use of an RSV G-specific antibody according to the invention, or a composition according to the invention, or a vector according to the invention, or a cell according to the invention, or an RSV bispecific antibody according to the invention for the preparation of a medicament and/or prophylactic agent for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder.
The invention further provides a method for at least in part treating and/or preventing an RSV infection and/or an RSV related disorder comprising administering to an individual, preferably a human, in need thereof a therapeutically effective amount of an RSV G-specific antibody according to the invention, and/or a nucleic acid sequence or functional equivalent thereof according to the invention, and/or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention, and/or a vector according to the invention, and/or a pharmaceutical composition according to the invention, and/or a cell according to the invention. In order to at least in part treat or prevent a disorder related to RSV, an RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, an RSV bispecific antibody, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell according to the invention is preferably administered to an individual before an RSV infection has taken place. Alternatively, an RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, an RSV bispecific antibody, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell according to the invention is administered when an individual is already infected. In that case, an RSV infection is counteracted, symptoms resulting from an RSV infection are alleviated and/or inflammation resulting from an RSV infection is counteracted. Said antibody, nucleic acid sequence, functional equivalent, composition, vector, pharmaceutical composition and/or cell is preferably administered to individuals with an increased risk of complications, such as hospitalized individuals, for instance infants, individuals with compromised immunity and/or elderly people. An RSV G-specific antibody, a nucleic acid sequence or functional equivalent thereof, a composition comprising a combination of at least two RSV G-specific antibodies, a vector, a pharmaceutical composition and/or a cell according to the invention is preferably administered via one or more injections. Typical doses of administration of an RSV G-specific antibody according to the invention, or combinations of at least two thereo, or of an RSV bispecific antibody are between 0.1 and 10 mg per kg body weight. For prophylactic or therapeutic application RSV G-specific antibodies according to the invention or RSV bispecific antibodies according to the invention are preferably combined with a pharmaceutically acceptable carrier, diluent and/or excipient.
The invention further provides a pharmaceutical composition comprising an RSV G-specific antibody according to the invention or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention, and a pharmaceutical acceptable carrier, diluent and/or excipient. Also provided is a pharmaceutical composition comprising an RSV bispecific antibody according to the invention and a pharmaceutical composition comprising an RSV G-specific antibody according to the invention coupled to an antiviral agent, antimicrobial peptide or immunomodulatory molecule as described herein. Further provided is a pharmaceutical composition comprising a nucleic acid sequence or functional equivalent according to the invention, or a vector or a cell according to the invention comprising such nucleic acid or functional equivalent, and a pharmaceutical acceptable carrier, diluent and/or excipient. Examples of suitable carriers for instance comprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin. In one preferred embodiment said suitable carrier comprises a solution, like for example saline. A pharmaceutical composition according to the invention is preferably suitable for human use. In one embodiment said pharmaceutical composition further comprises at least one other RSV specific antibody, preferably an RSV F protein specific antibody such as palivizumab, D25, AM14, AM16, AM22 and/or AM23.
An RSV G-specific antibody according to the present invention is also particularly suitable for diagnostic uses. For instance, if an individual, preferably a human, is suspected of suffering from an RSV infection, a sample, such as a saliva, sputum, blood, or tissue sample, can be obtained from said individual. Subsequently, said sample can be tested for the presence of G protein of RSV, using an RSV G-specific antibody according to the invention. Preferably, said sample is mixed with an RSV G-specific antibody according to the invention, which will specifically bind to a G protein of RSV. The presence of G proteins of RSV in a sample is indicative for the presence of an RSV infection. G proteins of RSV and/or RSV comprising a G protein bound to an RSV G-protein according to the invention can be isolated from the sample and/or detected using any method known in the art, for example, but not limited to, isolation using magnetic beads, streptavidin-coated beads, or isolation through the use of secondary antibodies immobilized on a column. Alternatively, or additionally, an RSV G-specific antibody according to the invention is labeled in order to be able to detect said antibody, for instance, but not limited to, fluorescently labeled, or radioactively labeled. Alternatively, an RSV G-specific antibody according to the invention is detected using a labeled secondary antibody which is directed against said antibody. If binding of said antibody is detected, G protein of RSV is present, which is indicative for the presence of an RSV infection. The invention thus provides an RSV G-specific antibody according to the invention, or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention for use in diagnosis of an RSV infection.
The invention thus further provides a method for determining whether an RSV G protein is present in a sample comprising:                contacting said sample with an RSV G-specific antibody according to the invention, or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention,        allowing said antibody or an antibody component of said composition to bind said RSV G protein, if present, and        determining whether RSV G protein is bound to said antibody, or to an antibody component of said composition, thereby determining whether an RSV G protein is present.        
In a preferred embodiment it is determined whether an individual is suffering from an RSV infection. Provided is therefore a method for determining whether an individual is suffering from an RSV infection comprising:                contacting a sample from said individual with an RSV G-specific antibody according to the invention, or a composition comprising a combination of at least two RSV G-specific antibodies according to the invention,        allowing said antibody, or an antibody component of said composition to bind said RSV, if present, and        determining whether RSV is bound to said antibody, or to an antibody component of said composition, thereby determining whether said individual is suffering from an RSV infection. Preferably said individual is a human.        
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
TABLE 1Preferred RSV G-specific antibodies according to the inventionSEQID NOAntibodyIdentitySequence  1AT46Heavy chain CDR1SRYVMS   2AT32Heavy chain CDR1ELSIH   3AT33Heavy chain CDR1SLAIS   4AT34Heavy chain CDR1HYGMH   5AT35Heavy chain CDR1TYWVS   6AT36Heavy chain CDR1YNFIDHSVS   7AT37Heavy chain CDR1SGGYSWN   8AT39Heavy chain CDR1TYAVH   9AT40Heavy chain CDR1DRHALH  10AT42Heavy chain CDR1SNVYYWG  11AT43Heavy chain CDR1NYGVS  12AT44Heavy chain CDR1SGHYWA  13AT45Heavy chain CDR1GHAIS  14AT47Heavy chain CDR1NYGIC  15AT49Heavy chain CDR1SLALN  16AT50Heavy chain CDR1NYGIS  17AT51Heavy chain CDR1KYGIN  18AM22Heavy chain CDR1KLSIH  19AT46Heavy chain CDR2SITGSGATTYYADSVKGRFTIS  20AT32Heavy chain CDR2GFEPEDGEYIYPQKSQG  21AT33Heavy chain CDR2GIIPKFNRRDYAQKFQG  22AT34Heavy chain CDR2VISYDGDKKYYADSVKG  23AT35Heavy chain CDR2NINQDGSEKSYVDSVEG  24AT36Heavy chain CDR2WISPYNHRTVYAEKFQG  25AT37Heavy chain CDR2YIYQNDITYYNPSLMS  26AT39Heavy chain CDR2WINPDNGDTKYSQRFQGRVVIT  27AT40Heavy chain CDR2ILSYDGTTDYYADSVKG  28AT42Heavy chain CDR2SIFHSGITHYTPSLNS  29AT43Heavy chain CDR2WISTYNGNTWYSQKFQA  30AT44Heavy chain CDR2GIHHSGSTYTNPPLKS  31AT45Heavy chain CDR2GIIPGLGTTRYARKFQD  32AT47Heavy chain CDR2WISGYNGNTYYAQNFQG  33AT49Heavy chain CDR2GIIPLFGTQNYAQKFQG  34AT50Heavy chain CDR2WISAYNGNTYYRQELQG  35AT51Heavy chain CDR2WISAYNGNTYYAQKFQG  36AM22Heavy chain CDR2GYEGEVDEIFYAQKFQ  37AT46Heavy chain CDR3CGRAGQIFDD  38AT32Heavy chain CDR3EARYCDNSRCSPNFDH  39AT33Heavy chain CDR3DAEWAAGSDYFFDY  40AT34Heavy chain CDR3QGAKGGHELSFYCALDV  41AT35Heavy chain CDR3EVFVTQVEPAQWGF  42AT36Heavy chain CDR3DRVQQGEGNFFDH  43AT37Heavy chain CDR3GAYGSGTYYSADALDI  44AT39Heavy chain CDR3GRIFDI  45AT40Heavy chain CDR3GRALDDFADYGGYYFDY  46AT42Heavy chain CDR3HAVAGLYFDS  47AT43Heavy chain CDR3HGSGNYYGEANYFDH  48AT44Heavy chain CDR3DLYDLSTGPFWFDP  49AT45Heavy chain CDR3VAGGYFDSATRG  50AT47Heavy chain CDR3GFHYHSADQRIFDP  51AT49Heavy chain CDR3FLWFGDQTSDDGFDV  52AT50Heavy chain CDR3GGAQEMVRIHYYYYGMDV  53AT51Heavy chain CDR3PATSYDDLRSGYLNYCDY  54AM22Heavy chain CDR3LGVTVTEAGLGIDDY  55AT46Light chain CDR1TLSSGHRNYAIA  56AT32Light chain CDR1KSSQSVLYDSNNKNYLA  57AT33Light chain CDR1SADAFSDQYAY  58AT34Light chain CDR1RASQGIGSWLA  59AT35Light chain CDR1RASQSIDNYLN  60AT36Light chain CDR1KSSQSLLHSSNNKIYLA  61AT37Light chain CDR1RASQSVSASNLA  62AT39Light chain CDR1QASQDISNFLN  63AT40Light chain CDR1RASQGISTWLA  64AT42Light chain CDR1RASQTVSSSHLA  65AT43Light chain CDR1RASESVSRNYLA  66AT44Light chain CDR1RASQSVSTKVV  67AT45Light chain CDR1RSSQSLLHSNGYNYLD  68AT47Light chain CDR1RASESISTWLA  69AT49Light chain CDR1RSSQSLLHGNGYKYLH  70AT50Light chain CDR1RASQVISSYLA  71AT51Light chain CDR1RASQGITSYLA  72AM22Light chain CDR1RASQIVSRNHLA  73AT46Light chain CDR2TNGSHYPGD  74AT32Light chain CDR2WASTRES  75AT33Light chain CDR2KDTERPS  76AT34Light chain CDR2NASGLES  77AT35Light chain CDR2LASTLQS  78AT36Light chain CDR2WASTRES  79AT37Light chain CDR2GASRTAT  80AT39Light chain CDR2DASKLQT  81AT40Light chain CDR2SASRLQS  82AT42Light chain CDR2GSSSRAT  83AT43Light chain CDR2GASSRAI  84AT44Light chain CDR2GASTRAT  85AT45Light chain CDR2GSNRAP  86AT47Light chain CDR2KASSLES  87AT49Light chain CDR2LGSNRAS  88AT50Light chain CDR2GASTLQT  89AT51Light chain CDR2AASTLQS  90AM22Light chain CDR2GASSRAT  91AT46Light chain CDR3QTWGAGI  92AT32Light chain CDR3QQYYDPL  93AT33Light chain CDR3QSTDTSGPL  94AT34Light chain CDR3QQYNSHT  95AT35Light chain CDR3QQSHSSP  96AT36Light chain CDR3QQYYTTHP  97AT37Light chain CDR3QQYGSSP  98AT39Light chain CDR3QKFDNLL  99AT40Light chain CDR3QQANTFP 100AT42Light chain CDR3QYYGDSP 101AT43Light chain CDR3QQYTIFP 102AT44Light chain CDR3QQYNKWP 103AT45Light chain CDR3MQALQTP 104AT47Light chain CDR3QQYKSYP 105AT49Light chain CDR3MQALQSP 106AT50Light chain CDR3QQLNTYP 107AT51Light chain CDR3QQFHTYP 108AM22Light chain CDR3LSSDSSI 109AT46Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYVMSWVRQAPGRGLEWVSSITGSGATTYYADSVKGRFTISRDNSKNTVYLQMNRLRAEDTAIYYCANCGRAGQIFDDWGQGTLVTVSS 110AT32Heavy chainQVQLVQSGAEMKKPGASVKVSCQVAGYTLTELSIHAVVRQTPGNGLEWMGGFEPEDGEYIYPQKSQGRVTMTEDTSTGTAYMELRSLRSDDTAVYYCAAEARYCDNSRCSPNFDHWGQGTLVAVSS 111AT33Heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGDSFNSLAISWVRQAPGQGLEWMGGIIPKFNRRDYAQKFQGRVTITADDSASTAYIELSSLTSDDTALYYCARDAEWAAGSDYFFDYWGQGTLVIVSS 112AT34Heavy chainQVQLMESGGGVVQPGKSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVAVISYDGDKKYYADSVKGRFTISRDNSKNTLHLHMNSLRHEDTAVYFCASQGAKGGHELSFYCALDVWGQGTTVAVSS 113AT35Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWVSWVRQTPGKGLEWVARFTISNINQDGSEKSYVDSVEGRDNAKNSLYLQMNSLRADDTAVYYCAREVFVTQVEPAQWGFWGQGTPVIVSS 114AT36Heavy chainQVQVVQSGAEVKKPGASVKVSCKTSGYNFIDHSVSWVRQAPGQGLEWMGWISPYNHRTVYAEKFQGRVTMTTDTSTRTVSMELRRLTSDDTAVYFCARDRVQQGEGNFFDHWGQGTPVTVTSA 115AT37Heavy chainQLQLQESGSRLVKPSQTLSLTCGVSGGSISSGGYSWNWIRQPPGKGLEWVGYIYQNDITYYNPSLMSRVTISADTSKNQFSLKLSSVTAADTAVYYCARGAYGSGTYYSADALDIWGQGTMVTVSS 116AT39Heavy chainQVQLVQSGPEVKKPGASVRLSCTASGNTFRTYAVHWVRQASGQRLEWMGWINPDNGDTKYSQRFQGRVVITRDTSARIIYLDLSSLTSEDTAVFYCFSGRIFDIWGQGTTITVSS 117AT40Heavy chainQVQLVESGGGVVQPGMSHRLSCAASTLIFDRHALHWVRQAPGAGLEWVAILSYDGTTDYYADSVKGRFTVSRDTSKNTVFLQMNGLRPQDTAVYYCARGRALDDFADYGGYYFDYWGQGILVTVSS 118AT42Heavy chainQVQLQESGPGLVQPSETLSLTCTVSGDSITSNGWIRQPPGKGLEWIGSIFHSGITHYTPSLNSRVTISVDTSKNQFSLRLSSATAADTAVYYCARHWAGLYFDSWGQGALVAVSS 119AT43Heavy chainQVQVVQSGPEVKKPGASVRVSCKASGYTFTNYGVSWVRQAPGQGLEWMGWISTYNGNTWYSQKFQARVTMTTDTSTSTAYMEVRSLRSDDTAIYYCACHGSGNYYGEANYFDHWGQGTLVTVSS 120AT44Heavy chainQVQLQASGPGLVKPSETLSLTCNVSGYSVSSGHYWAWVRQSPGKGLEWIGGIHHSGSTYTNPPLKSRVSISIDTSKNQFSLRLTSVTAADTAVYFCARDLYDLSTGPFWFDPWGQGTLVTVSS 121AT45Heavy chainQVHLVQSGAEVKKPGSSVKVSCKASGGTFNGHAISWIRQAPGQGLEWKGGIIPGLGTTRYARKFQDRVTITADESTRTAYMELSSLRSEDTAVYYCARVAGGYFDSATRGWGQGTLVTVSS 122AT47Heavy chainQVQLVQSGGEVKKPGASVKVSCKASGYTFTNYGICWVRQAPGQGLEWMGWISGYNGNTYYAQNFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGFHYHSADQRIFDPWGQGTLVTVSS 123AT49Heavy chainQVLLVQSGAEIKKPGSSVKISCKASGGTFSSLALNWVRQAPGQGLQWMGGIIPLFGTQNYAQKFQGRVTITADESTSTAYMELSGLRPEDTAVYYCALFLWFGDQTSDDGFDWGQGTVVTVSS 124AT50Heavy chainQVQLVQSGTEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLEWMGWISAYNGNTYYRQELQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGAQEMVRIHYYYYGMDWGQGTTVTVSS 125AT51Heavy chainQVQLVQSGAEVKKPGASMTVSCKASGYTFSKYGINWVRQAPGQGLEWLGWISAYNGNTYYAQKFQGRVTMTTDTATSTAYMDVRNLRSDDTAMYYCARPATSYDDLRSGYLNYCDYWGQGTLVTVSS 126AM22Heavy chainQVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGGYEGEVDEIFYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGIDDYWGQGTLVTVSS 127AT46Light chainQPVLTQSPSASASLGASVKLTCTLSSGHRNYAIAWHQQRPEKGPRYLMKIYTNGSHYPGDGTPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGAGIWVFGGGTKLTVLGQPK 128AT32Light chainDIVMTQSPDSLAVSLGERATFSCKSSQSVLYDSNNKNYLAWYQQRPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCQQYYDPLITFGQGTRLEIKRTV 129AT33Light chainSYELTQPPSVSVSPGQTARITCSADAFSDQYAYWYQQKPGQAPVLVIYKDTERPSGIPERISGSSSGTTATLSISGVQAEDEADYYCQSTDTSGPLFGGGTKLTLLGQPK 130AT34Light chainDIQMTQSPSTLSASVGDRVTITCRASQGIGSWLAWYQQKPGKAPKLLIYNASGLESGVPSGFSGSGSGTEFTLTISSLQPDDSATYYCQQYNSHTWTFGQGTKVEFKRTV 131AT35Light chainAIQMTQSPSSLSASVGDRVTISCRASQSIDNYLNWYQQKPGKAPKLLLFLASTLQSGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCQQSHSSPYSFGQGTKLEIKRTV 132AT36Light chainDIVMTQSPDSLAVSLGERATINCKSSQSLLHSSNNKIYLAWYQQKPGQPPKLLLYWASTRESGVPDRFTGSGSGTDFTLTINSLQAEDVAVYYCQQYYTTHPTFGQGTRLEIKRTV 133AT37Light chainKIVLTQSPGTLSLSPGERATLSCRASQSVSASNLAWYQQKPGQAPRLLIYGIPDRFSGSGSGTDFTLSISRLEPEDFAVYYCGASRTATQQYGSSPLTFGGGTKVEIKRTV 134AT39Light chainDIQMTQSPSSLSASVGDRVTITCQASQDISNFLNWYQQKPGQAPKLLIYDASKLQTGVPSRFSGSGSETDFTFTISSLQPEDVATYYCQKFDNLLLTFGGGTKVELKRTV 135AT40Light chainDIQMTQSPSSVSASVGDKVTITCRASQGISTWLAWYQQKPGKAPALLIYSASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQANTFPFTFGPGTKVDIKRTV 136AT42Light chainEIVLTQSPGTLSLSPGERATLSCRASQTVSSSHLAWYQQKPGQAPRLLIHGSSSRATGIPERFSGSGSGPDFTLTISRLKPEDFAVYYCQYYGDSPGSFGEGTKVEIKRTV 137AT43Light chainDIVLTQSPGTLSLSPGEGATLSCRASESVSRNYLAWYQQKPGQAPRLLIYGASSRAIGIPDRFSGSGSGTDFTLTISRLEPEDFAVYCCQQYTIFPLTFGGGTKVEIKRTV 138AT44Light chainEIVMTQSPATLSVSPGERVTLSCRASQSVSTKVVWYQQKFGQAPRLLIYGASTRATGIPVRFSGSGSGTEFTLTISSLQSEDLAVYFCQQYNKWPMYTFGQGTKLEIKRTV 139AT45Light chainDIVMTQSPLSLPVTPGESASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRAPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGQGTKVEIKRTV 140AT47Light chainDIQMTQSPSTLSASVGDRVTITCRASESISTWLAWYQQKPGKAPNLLIYKASSLESGVPSRFSGSGSGTEFTLAISSLQPDDFATYYCQQYKSYPYTFGQGTKLELKRTV 141AT49Light chainDIVMTQSPLSLTVTPGEPASISCRSSQSLLHGNGYKYLHWYLQKPGQSPQLLIYLGSNRASGVPARFSGSGSDTDFTLKISTVETEDVGVYYCMQALQSPTFGQGTKVEIKRTV 142AT50Light chainDIQLTQSPSFLSASVGDRVTITCRASQVISSYLAWYQQTPGRAPKLLIYGASTLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQLNTYPLTFGPGTKVEIKRTV 143AT51Light chainDIQLTQSPSFLSASVGDRVTITCRASQGITSYLAWYQQKPGRAPKLLIYAASTLQSGVASRFSGSGSGTEFTLTISSLQPEDFATYYCQQFHTYPLTFGGGTKVEIKRTV 144AM22Light chainEIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGASSRATGIPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKVDFK 145AT46Heavy chain CDR1agt aga tat gtc atg agt 146AT32Heavy chain CDR1gaa tta tcc ata cac 147AT33Heavy chain CDR1agt ctt gcc atc agt 148AT34Heavy chain CDR1cat tat ggc atg cac 149AT35Heavy chain CDR1acc tat tgg gtg agc 150AT36Heavy chain CDR1tac aac ttt atc gac cat agt gtc agc 151AT37Heavy chain CDR1agt ggt ggt tac tcc tgg aac 152AT39Heavy chain CDR1acc tat gct gta cat 153AT40Heavy chain CDR1gat aga cat gct ctc cac 154AT42Heavy chain CDR1agt aat gtt tac tac tgg ggc 155AT43Heavy chain CDR1aac tat ggt gtc agc 156AT44Heavy chain CDR1agc ggt cac tac tgg gcc 157AT45Heavy chain CDR1ggc cat gct atc agc 158AT47Heavy chain CDR1aac tac ggt atc tgt 159AT49Heavy chain CDR1agc ctt gct ctc aat 160AT50Heavy chain CDR1aac tat ggt atc agt 161AT51Heavy chain CDR1aag tat ggc atc aac 162AM22Heavy chain CDR1aaa tta tcc att cac 163AT46Heavy chain CDR2agc att act gga agt ggt gct acg aca tac tat gca gac tcc gtg aagggc cgc ttc acc atc tcc 164AT32Heavy chain CDR2ggt ttt gag cct gag gat ggt gag tac atc tac cca cag aaa tcc cagggc 165AT33Heavy chain CDR2ggg atc atc cct aag ttc aat aga aga gac tac gca cag aag ttt cagggc 166AT34Heavy chain CDR2gtc ata tcc tat gat ggc gat aaa aaa tat tat gca gac tca gtg aagggc 167AT35Heavy chain CDR2aac att aac caa gat gga agt gag aag tcc tat gtg gac tct gtg gagggc cga ttc acc atc tcc 168AT36Heavy chain CDR2tgg atc agc cct tac aac cac aga aca gta tat gca gag aag ttc cagggc 169AT37Heavy chain CDR2tac atc tat cag aat gac atc acc tac tac aac ccg tcc ctc atg agt 170AT39Heavy chain CDR2tgg atc aac cct gac aat ggt gac aca aaa tat tca cag agg ttc cagggt aga gtc gtc att acc 171AT40Heavy chain CDR2att ctc tct tat gat ggg acc aca gac tac tac gca gac tcc gtg aagggc 172AT42Heavy chain CDR2agt atc ttt cat agt ggg atc acc cac tat acc ccg tcc ctc aat agt 173AT43Heavy chain CDR2tgg atc agc act tac aat ggt aac aca tgg tat tca cag aag ttc caggcc 174AT44Heavy chain CDR2ggt atc cat cat agt ggg agt acc tac acc aat ccg ccc ctc aag agc 175AT45Heavy chain CDR2ggg atc atc cct ggc ctt ggt aca aca agg tac gca cgg aag ttc caggac 176AT47Heavy chain CDR2tgg atc agc ggt tac aat ggt aac aca tac tat gca cag aac ttc cagggc 177AT49Heavy chain CDR2ggg atc atc cct ctc ttt ggc act caa aac tac gca cag aag ttc cagggc 178AT50Heavy chain CDR2tgg atc agc gct tac aat ggt aac aca tac tat aga cag gag ctc cagggc 179AT51Heavy chain CDR2tgg atc agc gca tac aat ggc aac aca tac tat gca cag aag ttc cagggc 180AM22Heavy chain CDR2ggt tat gag ggt gag gtc gat gag att ttc tac gca cag aag ttc cagcac 181AT46Heavy chain CDR3tgt ggt agg gcg ggc caa att ttt gac gac 182AT32Heavy chain CDR3gag gca aga tat tgt gat aac agc aga tgt tcc cct aac ttt gac cac 183AT33Heavy chain CDR3gac gcc gag tgg gca gct ggc tcg gat tac ttc ttt gac tac 184AT34Heavy chain CDR3cag ggg gca aag ggc ggt cac gaa ctt tct ttc tac tgt gct ttg gacgtc 185AT35Heavy chain CDR3gaa gtc ttc gtg act cag gtg gag ccc gcg cag tgg ggc ttc 186AT36Heavy chain CDR3gat cga gta caa cag ggc gag gga aac ttc ttt gac cac 187AT37Heavy chain CDR3ggg gcc tat ggt tcg gga act tat tat tcc gct gat gct ctt gat ata 188AT39Heavy chain CDR3ggg aga att ttt gat ata 189AT40Heavy chain CDR3gga agg gcc cta gat gac ttc gct gac tac ggg gga tac tac ttt gactac 190AT42Heavy chain CDR3cat tgg gct ggc ctc tac ttt gac tct 191AT43Heavy chain CDR3cac ggg agt ggc aat tac tac ggc gaa gcg aac tac ttt gac cac 192AT44Heavy chain CDR3gat ctg tac gat ctt tcg acg ggg cct ttt tgg ttc gac ccc 193AT45Heavy chain CDR3gtg gcc ggg gga tac ttc gat agt gct act cga ggc 194AT47Heavy chain CDR3ggg ttt cac tat cat agt gct gat cag aga ata ttc gac ccc 195AT49Heavy chain CDR3ttt ctt tgg ttc ggg gac caa acg agt gat gat ggt ttt gat gtc 196AT50Heavy chain CDR3ggg ggt gcc caa gag atg gtt aga ata cac tac tac tac tac gga atggac gtc 197AT51Heavy chain CDR3ccc gca acc tca tat gac gat ctt cgg agt ggt tat ttg aac tac tgtgac tac 198AM22Heavy chain CDR3cta ggt gtg aca gtg act gag gct gga ctg ggg atc gat gac tac 199AT46Light chain CDR1act ctg agc agt ggg cac agg aac tac gcc atc gca 200AT32Light chain CDR1aag tcc agc cag agt gtt tta tac gac tcc aac aat aag aac tac ttagct 201AT33Light chain CDR1tct gca gat gca ttt tca gac caa tat gct tat 202AT34Light chain CDR1cgg gcc agt cag ggt att ggt agt tgg ttg gcc 203AT35Light chain CDR1cgg gca agt cag agc att gac aac tat tta aat 204AT36Light chain CDR1aag tcc agc cag agt ctt tta cac agc tcc aac aat aag atc tac ttagct 205AT37Light chain CDR1agg gcc agt cag agt gtt agc gcc agc aac tta gcc 206AT39Light chain CDR1cag gcg agt cag gac att agc aac ttt tta aat 207AT40Light chain CDR1cgg gcg agt cag ggt att agt acc tgg tta gcc 208AT42Light chain CDR1agg gcc agt cag act gta agc agc agc cac tta gcc 209AT43Light chain CDR1agg gcc agt gag agt gtt agc cgc aac tac tta gcc 210AT44Light chain CDR1agg gcc agt cag agt gtc agc acc aag gta gtc 211AT45Light chain CDR1agg tct agt cag agc ctc ctg cat agt aat gga tac aac tat ttg gat 212AT47Light chain CDR1cgg gcc agt gag agt att agt acc tgg ttg gcc 213AT49Light chain CDR1agg tct agt cag agc ctc ctg cat ggt aat gga tac aaa tat ctg cac 214AT50Light chain CDR1cgg gca agc cag gtc att agc agt tat tta gcc 215AT51Light chain CDR1cgg gca agt cag ggc att acc agt tat tta gcc 216AM22Light chain CDR1agg gcc agt cag att gtt agc agg aac cac tta gcc 217AT46Light chain CDR2act aat ggc agc cac tac ccg ggg gac 218AT32Light chain CDR2tgg gcg tct acc cgg gaa tcc 219AT33Light chain CDR2aaa gac act gag agg ccc tca 220AT34Light chain CDR2aac gcg tct ggc tta gaa agt 221AT35Light chain CDR2ctt gcg tcc act ttg caa agt 222AT36Light chain CDR2tgg gca tct acc cgg gag tcc 223AT37Light chain CDR2ggt gca tcc agg acg gcc act 224AT39Light chain CDR2gat gcg tcc aaa ttg caa aca 225AT40Light chain CDR2tct gca tcc aga ttg cag agt 226AT42Light chain CDR2ggt tca tct agc agg gcc aca 227AT43Light chain CDR2ggt gca tcc agc agg gcc att 228AT44Light chain CDR2ggt gca tcc acc agg gcc act 229AT45Light chain CDR2ggt tct aat cgg gcc ccc 230AT47Light chain CDR2aag gcg tct agt tta gaa agt 231AT49Light chain CDR2ttg ggt tct aat cgg gcc tcc 232AT50Light chain CDR2ggt gca tcc acg tta caa act 233AT51Light chain CDR2gct gca tcc act ttg caa agt 234AM22Light chain CDR2ggt gcg tcc agt cgg gcc act 235AT46Light chain CDR3cag acc tgg ggc gct ggc att 236AT32Light chain CDR3caa caa tat tat gat cct ctc 237AT33Light chain CDR3caa tca aca gac acc agt ggt cct tta 238AT34Light chain CDR3caa caa tac aat agt cac acg 239AT35Light chain CDR3caa cag agc cac tct tcc ccc 240AT36Light chain CDR3cag caa tat tat act act cat ccc 241AT37Light chain CDR3caa cag tat ggt agc tca ccg 242AT39Light chain CDR3caa aag ttt gat aat ctc ctt 243AT40Light chain CDR3caa cag gct aac act ttc ccc 244AT42Light chain CDR3cag tac tat ggt gac tca ccc 245AT43Light chain CDR3tgt cag cag tat act atc ttc cct 246AT44Light chain CDR3cag cag tat aat aag tgg ccc 247AT45Light chain CDR3atg caa gct cta caa act cct 248AT47Light chain CDR3caa cag tat aaa agt tac ccg 249AT49Light chain CDR3atg caa gct cta caa agt ccg 250AT50Light chain CDR3caa cag ctt aat act tac ccc 251AT51Light chain CDR3caa cag ttt cat act tac ccg 252AM22Light chain CDR3ctg tcc tct gat tcc tcc ata 253AT46Heavy chaingag gtg cag ctg gtg gag tct ggg gga ggc ttg gta cag cct ggg gggtcc ctg cga ctc tcc tgt gca gcc tct gga ttc acc ttt agt aga tatgtc atg agt tgg gtc cgc cag gct cca ggg agg ggc ctg gag tgg gtctca agc att act gga agt ggt gct acg aca tac tat gca gac tcc gtgaag ggc cgc ttc acc atc tcc aga gac aat tcc aag aac acg gtg tatctg caa atg aac agg ctg aga gcc gag gac acg gcc ata tat tac tgtgcg aat tgt ggt agg gcg ggc caa att ttt gac gac tgg ggc cag ggaacc ctg gtc acc gtc tcc tca 254AT32Heavy chaincag gtc cag ctg gta caa tct ggg gct gag atg aag aag cct ggg gcctca gtg aag gtc tcc tgc cag gtt gcc gga tac acc ctc act gaa ttatcc ata cac tgg gtg cga cag act cct gga aac ggg ctt gag tgg atggga ggt ttt gag cct gag gat ggt gag tac atc tac cca cag aaa tcccag ggc aga gtc acc atg acc gag gac aca tct aca ggc aca gcc tacatg gaa ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgtgca gcc gag gca aga tat tgt gat aac agc aga tgt tcc cct aac tttgac ac tgg ggc cag gga acc ctg gtc gcc gtc tcc tca 255AT33Heavy chaincag gtg cag ttg gtg cag tct ggg gct gag gtg aag aag cct ggg tcctcg gtg aag gtc tcc tgc aag gct tct gga gac tcc ttc aac agt cttgcc atc agt tgg gtg cga cag gcc cct gga caa gga ctc gag tgg atggga ggg atc atc cct aag ttc aat aga aga gac tac gca cag aag tttcag ggc aga gtc acg att acc gcg gac gac tcc gcg agc aca gcc tacata gag ttg agc agc ctg aca tct gac gac aca gcc ctg tat tac tgtgcg aga gac gcc gag tgg gca gct ggc tcg gat tac ttc ttt gac tactgg ggc cag gga acc ctg gtc atc gtc tcc tca 256AT34Heavy chaincag gtg caa ttg atg gag tct ggg gga ggc gtg gtc cag cct ggg aagtcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt cat tatggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtggca gtc ata tcc tat gat ggc gat aaa aaa tat tat gca gac tca gtgaag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg catctc cac atg aat agc ctg aga cat gag gac acg gct gtc tat ttc tgtgcc tcc cag ggg gca aag ggc ggt cac gaa ctt tct ttc tac tgt gctttg gac gtc tgg ggc caa ggg acc acg gtc gcc gtc tcc tca 257AT35Heavy chaingag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag ccg ggg gggtcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt agt acc tattgg gtg agc tgg gtc cgc cag act cca ggg aag gga ctg gag tgg gtggcc aac att aac caa gat gga agt gag aag tcc tat gtg gac tct gtggag ggc cga ttc acc atc tcc aga gac aac gct aag aac tcg ctg tatctg caa atg aac agc ctg aga gcc gac gac acg gct gta tat tat tgtgcg aga gaa gtc ttc gtg act cag gtg gag ccc gcg cag tgg ggc ttctgg ggc cag gga acc ccg gtc atc gtc tcc tcc 258AT36Heavy chaincag gtt cag gtg gtg cag tct gga gcc gag gtg aag aag cct ggg gcctca gtc aag gtc tct tgc aag act tct ggt tac aac ttt atc gac catagt gtc agc tgg gtg cga cag gcc ccc ggc caa ggg ctt gag tgg atggga tgg atc agc cct tac aac cac aga aca gta tat gca gag aag ttccag ggc aga gtc acc atg acc aca gac aca tcg acg agg aca gtc tccatg gag ttg agg agg ctg aca tct gac gac acg gcc gtc tac ttc tgtgcg cga gat cga gta caa cag ggc gag gga aac ttc ttt gac cac tggggc cag gga acc ccg gtc acc gtc acc tca gcc 259AT37Heavy chaincag ctg cag ctg cag gag tcc ggc tcc aga ctg gtg aag cct tca cagacc ctg tcc ctc acc tgc ggt gtc tct ggt ggc tcc atc agc agt ggtggt tac tcc tgg aac tgg atc cgg cag cca cca ggg aag ggc ctg gagtgg gtt ggg tac atc tat cag aat gac atc acc tac tac aac ccg tccctc atg agt cga gtc acc ata tca gca gac acg tcc aag aac cag ttctcc ctg aag ttg agc tct gtg acc gcc gcg gac acg gcc gtg tat tactgt gcc cga ggg gcc tat ggt tcg gga act tat tat tcc gct gat gctctt gat ata tgg ggc caa ggg aca atg gtc acc gtc tct tca 260AT39Heavy chaincag gtc cag ctt gtg cag tct ggg cct gag gtg aag aag cct ggg gcctca gtg agg ctt tcc tgt acg gcc tct gga aac acc ttc cgt acc tatgct gta cat tgg gtg cgc cag gcc tcc gga caa aga ctt gag tgg atggga tgg atc aac cct gac aat ggt gac aca aaa tat tca cag agg ttccag ggt aga gtc gtc att acc agg gac aca tcc gcg agg ata atc tacttg gac ctg agc agc ctg aca tct gaa gac acg gct gtg ttc tat tgtttc agc ggg aga att ttt gat ata tgg ggc caa ggg aca acg atc accgtc tct tca 261AT40Heavy chaincag gtg cag ctg gtg gag tcc ggg gga ggc gtg gtc cag cct ggg atgtcc cac aga ctc tcc tgt gca gcc tct aca ttg atc ttc gat aga catgct ctc cac tgg gtc cgc cag gct cca ggc gcg ggc ctg gag tgg gtggcg att ctc tct tat gat ggg acc aca gac tac tac gca gac tcc gtgaag ggc cga ttc acc gtc tcc aga gac acc tcc aag aac aca gtg tttcta caa atg aac ggc ctg aga cct caa gac acg gct gtt tat tac tgtgcg aga gga agg gcc cta gat gac ttc gct gac tac ggg gga tac tacttt gac tac tgg ggc cag gga atc ctg gtc acc gtc tcc tca 262AT42Heavy chaincag gtg cag ctg cag gag tcc ggc cca gga ctg gtg cag cct tcg gagacc ctg tcc ctc act tgc act gtt tct ggt gac tcc atc acc agt aatgtt tac tac tgg ggc tgg atc cgc cag ccc cca ggg aag ggg ctg gagtgg att ggg agt atc ttt cat agt ggg atc acc cac tat acc ccg tccctc aat agt cga gtc acc ata tcc gtc gac acg tcc aag aac cag ttctcc ctg aga ctg agt tct gcg acc gcc gca gac acg gct gta tat tattgt gcg agg cat tgg gct ggc ctc tac ttt gac tct tgg ggc cag ggagcc ctg gtc gcc gtc tcc tca 263AT43Heavy chaincag gtt cag gtg gtg cag tct gga cct gag gtg aag aag cct ggg gcctca gtg agg gtc tcc tgc aag gct tct ggt tac acc ttt acc aac tatggt gtc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atggga tgg atc agc act tac aat ggt aac aca tgg tat tca cag aag ttccag gcc aga gtc acc atg acc aca gac act tcc acg agc aca gcc tacatg gag gtg agg agc ctg aga tct gac gac acg gcc ata tat tac tgtgcg tgc cac ggg agt ggc aat tac tac ggc gaa gcg aac tac ttt gaccac tgg ggc cag gga acc ctg gtc acc gtc tcc tcc 264AT44Heavy chaincag gtg cag ctg cag gcg tcg ggc cca gga ctg gtg aag cct tcg gagacc ctg tcc ctc acc tgt aat gtc tct ggc tac tcc gtc agt agc ggtcac tac tgg gcc tgg gtc cgg cag tcc cca ggg aag ggg ctg gag tggatt ggg ggt atc cat cat agt ggg agt acc tac acc aat ccg ccc ctcaag agc cga gtc tcc ata tca ata gac acg tcc aag aac cag ttc tctttg agg ttg acc tct gtg acc gcc gca gac acg gcc gtg tat ttc tgtgcg aga gat ctg tac gat ctt tcg acg ggg cct ttt tgg ttc gac ccctgg ggc cag gga acc ctg gtc acc gtc tcc tca 265AT45Heavy chaincag gtg cac ctg gtg cag tct ggg gct gag gtg aag aag cct ggg tcctcg gtg aag gtc tcc tgc aag gct tct gga ggc acc ttc aac ggc catgct atc agc tgg ata cga cag gcc cct gga caa gga ctt gag tgg aaggga ggg atc atc cct ggc ctt ggt aca aca agg tac gca cgg aag ttccag gac aga gtc acg att acc gcg gac gaa tcc acg agg aca gcc tacatg gag ctg agc agc ctg aga tct gag gac acg gcc gtc tat tac tgtgcg aga gtg gcc ggg gga tac ttc gat agt gct act cga ggc tgg ggccag gga acc ctg gtc acc gtc tcc tca 266AT47Heavy chaincag gtt cag ctg gtg cag tct gga ggt gag gtg aag aag cct ggg gcctca gtg aag gtc tcc tgt aag gct tct ggt tac acc ttt acc aac tacggt atc tgt tgg gtg cga cag gcc cct gga caa ggg ctt gaa tgg atggga tgg atc agc ggt tac aat ggt aac aca tac tat gca cag aac ttccag ggc aga gtc acc atg acc aca gac aca tcc acg agc aca gcc tacatg gag ctg agg agc ctg aga tct gac gac acg gcc gta tat tac tgtgcg aga ggg ttt cac tat cat agt gct gat cag aga ata ttc gac ccctgg ggc cag gga acc ctg gtc acc gtc tcc tca 267AT49Heavy chaincag gtg ctt ctg gtg cag tct ggg gct gag ata aag aag cct ggg tcctcg gtg aaa atc tcc tgc aag gcc tct gga ggg acc ttc agc agc cttgct ctc aat tgg gtg cga cag gcc cct gga cag ggg ctt cag tgg atggga ggg atc atc cct ctc ttt ggc act caa aac tac gca cag aag ttccag ggc aga gtc acc att acc gcg gac gaa tcc acg agc aca gcc tacatg gag ctg agc ggc ctg cga ccc gag gac acg gcc gtc tat tac tgtgcc cta ttt ctt tgg ttc ggg gac caa acg agt gat gat ggt ttt gatgtc tgg ggc caa ggg aca gtg gtc acc gtg tct tca 268AT50Heavy chaincag gtt cag ctg gtg cag tct gga act gag gtg aag aag cct ggg gcctca gtg aag gtc tcc tgc aag gct tct ggt tac acc ttt agc aac tatggt atc agt tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atggga tgg atc agc gct tac aat ggt aac aca tac tat aga cag gag ctccag ggc aga gtc acc atg acc aca gac aca tcc acg agc aca gcc tacatg gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgtgcg aga ggg ggt gcc caa gag atg gtt aga ata cac tac tac tac tacgga atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 269AT51Heavy chaincag gtt cag ctg gtg cag tct gga gct gag gtg aag aag cct ggg gcctca atg acg gtc tcc tgc aag gcc tct ggt tac acc ttt tcc aag tatggc atc aac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg ctgggt tgg atc agc gca tac aat ggc aac aca tac tat gca cag aag ttccag ggc aga gtc acc atg acc aca gac aca gcc acg agc aca gcc tacatg gac gtg agg aac ctg aga tct gac gac acg gcc atg tat tac tgtgcg agg ccc gca acc tca tat gac gat ctt cgg agt ggt tat ttg aactac tgt gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 270AM22Heavy chaincag gtc cag ctg gta cag tct ggg gct gag gtg aag aag ccc ggg gccaca gtg aaa gtc tcc tgc aag att tcc gga cac acc ctc att aaa ttatcc att cac tgg gtg cga cag gct cct gga aag ggg ctt gag tgg atggga ggt tat gag ggt gag gtc gat gag att ttc tac gca cag aag ttccag cac aga ctc acc gtg atc gcc gac aca gcg aca gac aca gtc tacatg gaa ctg ggc agg ctc acc tct gac gac acg gcc gtc tat ttc tgtgga aca cta ggt gtg aca gtg act gag gct gga ctg ggg atc gat gactac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 271AT46Light chaincag cct gtg ctg act caa tcg ccc tct gcc tct gcc tcc ctg gga gcctcg gtc aag ctc acc tgc act ctg agc agt ggg cac agg aac tac gccatc gca tgg cat cag cag cga cca gag aag ggc cct cgt tac ttg atgaag att tat act aat ggc agc cac tac ccg ggg gac ggg acc cct gatcgc ttc tca ggc tcc agc tct ggg gct gag cgc tac ctc acc atc tccagc ctc caa tct gag gat gag gct gac tat tac tgt cag acc tgg ggcgct ggc att tgg gtt ttc ggc gga ggg acc aag ctg acc gtc cta ggtcag ccc aag 272AT32Light chaingac atc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg ggcgag agg gcc acc ttc agc tgc aag tcc agc cag agt gtt tta tac gactcc aac aat aag aac tac tta gct tgg tac cag cag aga cca gga cagcct cct aag ttg ctc att tac tgg gcg tct acc cgg gaa tcc ggg gtccct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc accatc agc agt ctg cag cct gaa gat gtg gca gtt tat tac tgt caa caatat tat gat cct ctc atc acc ttc ggc caa ggg aca cga ctg gag attaaa cga act gtg 273AT33Light chaintcc tat gag ctg act cag cca ccc tcg gtg tca gtg tcc cca gga cagacg gcc agg atc acc tgc tct gca gat gca ttt tca gac caa tat gcttat tgg tac cag cag aag cca ggc cag gcc cct gtg ttg gtg ata tataaa gac act gag agg ccc tca ggg atc cct gag cga atc tct ggc tccagc tca ggg aca aca gcc acg ttg agc atc agt gga gtc cag gca gaagac gag gct gac tat tac tgt caa tca aca gac acc agt ggt cct ttattc ggc gga ggg acg aag ctg acc ctc cta ggt cag ccc aag 274AT34Light chaingac atc cag atg acc cag tct cct tcc acc ctg tct gca tct gtg ggagac aga gtc acc atc act tgt cgg gcc agt cag ggt att ggt agt tggttg gcc tgg tat cag cag aaa cca ggg aaa gcc cca aaa ctc ctg atctat aac gcg tct ggc tta gaa agt ggc gtc cca tca ggg ttc agc ggcagt gga tct ggg aca gag ttc act ctc acc atc agc agc ctg cag cctgat gat tct gcg acg tat tac tgc caa caa tac aat agt cac acg tggaca ttc ggc caa ggg acc aag gtg gaa ttc aag cga act gtg 275AT35Light chaingcc atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta ggagac aga gtc acc atc tct tgc cgg gca agt cag agc att gac aac tattta aat tgg tat cag cag aaa ccg ggg aaa gcc cct aaa ctc ctg ctcttt ctt gcg tcc act ttg caa agt ggt gtc cct tca agg ttc act ggcagt gga tct ggg aca gat ttc act ctc acc atc agc agt ctt caa cctgaa gat ttt gcg act tac tac tgt caa cag agc cac tct tcc ccc tacagt ttt ggc cag ggg acc aag ctt gag atc aaa cga act gtg 276AT36Light chaingac atc gtg atg acc cag tct cca gac tct ctg gct gtg tct ctg ggcgag agg gcc acc atc aac tgc aag tcc agc cag agt ctt tta cac agctcc aac aat aag atc tac tta gct tgg tac cag cag aaa cca gga cagcct cct aag tta ctc ctt tac tgg gca tct acc cgg gag tcc ggg gtccct gac cgc ttc act ggc agc ggg tct ggg aca gat ttc act ctc accatc aac agc ctg cag gct gag gat gtg gct gtt tat tac tgt cag caatat tat act act cat ccc act ttt ggc cag ggg acc agg ctg gag atcaaa cga act gtg 277AT37Light chainaaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggggaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc gcc agcaac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctcatc tat ggt gca tcc agg acg gcc act ggc atc cca gac agg ttc agtggc agt ggg tct ggg aca gac ttc act ctc tcc atc agc aga ctg gagcct gaa gat ttt gca gtg tat tac tgt caa cag tat ggt agc tca ccgctc act ttc ggc gga ggg acc aag gtg gag atc aaa cga act gtg 278AT39Light chaingac atc cag atg acc cag tct cca tcc tcc ctg tca gca tct gtg ggagac aga gtc acc atc act tgc cag gcg agt cag gac att agc aac ttttta aat tgg tat cag cag aaa ccg ggc caa gcc cct aaa ctc ctg atctat gat gcg tcc aaa ttg caa aca ggg gtc ccg tca agg ttc agt ggaagt ggt tct gag aca gac ttt act ttc acc atc agc agc ctg cag cctgaa gat gtt gca aca tat tac tgt caa aag ttt gat aat ctc ctt ctcact ttc ggc gga ggg acc aag gtg gag ctc aag cga act gtg 279AT40Light chaingac atc cag atg acc cag tct cca tct tcc gta tct gcg tct gtg ggagac aaa gtc acc atc acc tgt cgg gcg agt cag ggt att agt acc tggtta gcc tgg tat cag cag aaa cct ggg aaa gct cct gcc ctc ctg atatat tct gca tcc aga ttg cag agt ggg gtc ccc tca agg ttt agc ggcagt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg cag cctgaa gat tat gca acc tat tat tgt caa cag gct aac act ttc ccc ttcact ttc ggc cct ggg acc aaa gtg gac atc aaa cga act gtg 280AT42Light chaingaa atc gtg ttg acg cag tct cca ggc acc ctg tct ctg tct cca ggggaa aga gcc acc ctc tcc tgc agg gcc agt cag act gta agc agc agccac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctcatc cat ggt tca tct agc agg gcc aca ggc atc cca gag agg ttc agtggc agt ggg tct ggg cca gac ttc act ctc acc atc tcc aga ctg aagcct gaa gat ttt gct gtg tat tac tgt cag tac tat ggt gac tca cccggc tct ttc ggc gaa ggg acc aag gtg gag atc aaa cga act gtg 281AT43Light chaingac att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggggaa gga gcc acc ctc tcc tgc agg gcc agt gag agt gtt agc cgc aactac tta gcc tgg tac cag caa aaa cct ggc cag gct ccc agg ctc ctcatc tat ggt gca tcc agc agg gcc att ggc atc cca gac agg ttc agtggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gagcct gaa gat ttt gca gta tac tgc tgt cag cag tat act atc ttc cctctc act ttc ggc gga ggg acc aag gtg gag atc aaa cga act gtg 282AT44Light chaingaa atc gtg atg acg cag tca cca gcc acc ctg tct gtg tct cca ggggag aga gtc acc ctc tcc tgt agg gcc agt cag agt gtc agc acc aaggta gtc tgg tac cag cag aaa ttt ggc cag gct ccc agg ctc ctc atctat ggt gca tcc acc agg gcc act ggt atc cca gtc agg ttc agt ggcagt ggg tct ggg aca gag ttc act ctc acc atc agc agc ctg cag tctgaa gat ctt gca gtt tat ttc tgt cag cag tat aat aag tgg ccc atgtac act ttt ggc cag ggg acc aag ttg gaa atc aaa cga act gtg 283AT45Light chaingat att gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct ggagag tcg gcc tcc atc tcc tgc agg tct agt cag agc ctc ctg cat agtaat gga tac aac tat ttg gat tgg tac ctg cag aag cca ggg cag tctcca cag ctc ctg atc tat ttg ggt tct aat cgg gcc ccc ggg gtc cctgac agg ttt agt ggc agt gga tca ggc aca gat ttt aca ctg aaa atcagc aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gctcta caa act cct acg ttc ggc caa ggg acc aag gtg gaa atc aaa cgaact gtg 284AT47Light chaingac atc cag atg acc cag tct cct tcc acc ctg tct gca tct gta ggagac aga gtc acc atc act tgc cgg gcc agt gag agt att agt acc tggttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aac ctc ctg atctat aag gcg tct agt tta gaa agt ggg gtc cca tca agg ttc agc ggcagt gga tct ggg aca gaa ttc act ctc gcc atc agc agc ctg cag cctgat gat ttt gca act tat tac tgc caa cag tat aaa agt tac ccg tacact ttt ggc cag ggg acc aag ctg gag ctg aaa cga act gtg 285AT49Light chaingat att gtg atg act cag tca ccg ctc tcc ctg acc gtc acc ccg ggagag ccg gcc tcc atc tca tgc agg tct agt cag agc ctc ctg cat ggtaat gga tac aaa tat ctg cac tgg tac ctg cag aag cca ggg cag tctcca cag ctc ctg atc tat ttg ggt tct aat cgg gcc tcc ggg gtc cctgcc agg ttc agt ggc agt gga tca gac aca gat ttt act ctg aaa atcagc acc gtg gag act gag gat gtt ggg gtt tat tac tgc atg caa gctcta caa agt ccg acg ttc ggc caa ggg act aag gtg gaa atc aaa cgaact gtg 286AT50Light chaingac atc cag ttg acc cag tct cca tcc ttc ctg tct gca tct gta ggagac aga gtc acc atc act tgc cgg gca agc cag gtc att agc agt tattta gcc tgg tat cag caa aca cca ggg aga gcc cct aag ctc ctg atctat ggt gca tcc acg tta caa act ggg gtc cca tca agg ttc agc ggcagt gga tct ggg aca gaa ttc act ctc aca atc agc agc ctg cag cctgaa gat ttc gca act tat ttc tgt caa cag ctt aat act tac ccc ctcact ttc ggc cct ggg acc aaa gtg gag atc aaa cga act gtg 287AT51Light chaingac atc cag ttg acc cag tct cca tcc ttc ctg tct gca tct gta ggagac aga gtc acc atc act tgc cgg gca agt cag ggc att acc agt tattta gcc tgg tat cag caa aaa cca ggg aga gcc cct aag ctc ctg atctat gct gca tcc act ttg caa agt ggg gtc gca tca agg ttc agc ggcagt gga tct ggg aca gaa ttc act ctc aca atc agc agc ctg cag cctgaa gat ttt gca act tat tac tgt caa cag ttt cat act tac ccg ctcact ttc ggc gga ggg acc aag gtg gag atc aaa cga act gtg 288AM22Light chaingaa att gtg ttg aca cag tct cca ggc acc ctg tct ttg tct cca ggagaa aga gcc acc ctc tcc tgc agg gcc agt cag att gtt agc agg aaccac tta gcc tgg tac cag caa aaa cct ggc cag gct ccc agg ctc ctcatc ttt ggt gcg tcc agt cgg gcc act ggc atc cca gtc cgg ttc agtggc agt ggg tct ggg aca gac ttc act ctc acc atc aac gga ctg gcgcct gaa gat ttt gca gtt tac tac tgt ctg tcc tct gat tcc tcc atattc aca ttc ggc cct ggg acc aag gtg gat ttc aaa
TABLE 2Preferred combinations of RSV G-specific antibodies according to the inventionAT43 + AT49AT43 + AT40AT51 + AT34AT51 + AT40AT47 + AT44AT47 + AT34AT47 + AT49AT35 + AT45AT35 + AT44AT35 + AT34AT35 + AT49AT35 + AT40AT37 + AT45AT37 + AT34AT37 + AT49AT37 + AT40AT39 + AT45AT39 + AT44AT39 + AT34AT39 + AT49AT39 + AT40AT32 + AT45AT32 + AT44AT32 + AT34AT32 + AT49AT32 + AT40AT32 + AT31AT33 + AT45AT33 + AT44AT33 + AT34AT33 + AT49AT33 + AT40AT33 + AT42AT33 + AT38AT33 + AT50AT33 + AT36AT33 + AT46AT42 + AT44AT46 + AT38AT46 + AT45AT46 + AT44AT46 + AT34AT46 + AT49AT46 + AT40AT36 + AT45AT36 + AT44AT36 + AT34AT36 + AT49AT50 + AT44AT50 + AT40AT31 + AT44AT31 + AT34
TABLE 3Particularly preferred combinations of RSV G-specific antibodies according to the inventionAT43 + AT49AT51 + AT34AT47 + AT34AT35 + AT45AT35 + AT44AT35 + AT34AT37 + AT45AT37 + AT34AT39 + AT45AT39 + AT34AT39 + AT49AT39 + AT40AT32 + AT45AT32 + AT44AT32 + AT34AT32 + AT40AT32 + AT31AT33 + AT45AT33 + AT44AT33 + AT34AT33 + AT49AT33 + AT40AT33 + AT42AT33 + AT38AT33 + AT50AT33 + AT46AT46 + AT45AT46 + AT44AT46 + AT34AT46 + AT40AT36 + AT45AT36 + AT34AT36 + AT49AT50 + AT44AT31 + AT34Table 4. Summary of preferred RSV G-specific antibodies according to the invention. Provided in FIG. 8.
TABLE 5Binding of B cell supernatants containing anti-RSV G IgG to RSV infected cells expressing native viral proteins, detected with anti-huIgG-PE.RSV XRSV 2007-2clone nameRSV A2subtype Asubtype BAT46+++AT42+++AT40+++AT44+++AT45+++AT49+++AT34+++AT32++negAT33++negAT35++negAT36++negAT37++negAT39++negAT43++negAT47++negAT50++negAT51++negpalivizumah+++rD25+++ctrl anti IgG-PEnegnegneg
TABLE 6Summary of antibody binding to RSV infectedcell lysates by ELISA and Western Blot.antibodycloneELISAWBAT46−−AT42−−AT40++AT44++AT45++AT49++AT34++AT32++AT33++AT35++AT36++AT37++AT39++AT43+/−+/−AT47+/−+/−AT50++AT51++
TABLE 7aka, kd and KD of antibodies AT40, AT44, AT32, AT42 and AT49 to RSV Ga. ka is indicated in 104 sec−1*M−1, kd in 10−4 sec−1, KD in nM. Constants were fitted in Scrubber2, using a global fit to all SPR curves.Antibody:ka:kd:KD (RSV A2 (G)):AT32 75 (±21)4.3 (±0.3)0.6 (±0.1) AT4035 (±2)0.6 (±0.1)0.2 (±0.01)AT42 64 (±11)7.6 (±1.7)1.3 (±0.4) AT4435 (±4)0.3 (±0.1)0.1 (±0.02)AT4922 (±3) 0.3 (±0.04)0.1 (±0.01)
TABLE 7bka, kd and KD of antibodies AT40, AT44, AT42 and AT49 to RSV Gb.Antibody:ka:kd:KD (RSV G (B1)):AT4034 (±10)0.3 (±0.15)0.1 (±0.07)AT4240 (±14)1.0 (±0.11)0.3 (±0.1) AT4434 (±16)0.4 (±0.17)0.1 (±0.07)AT4912 (±6) 0.5 (±0.02)0.5 (±0.2) 